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Preview: Molecular Human Reproduction - current issue

Molecular Human Reproduction - current issue

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Progress towards human primordial germ cell specification in vitro


Primordial germ cells (PGCs) have long been considered the link between one generation and the next. PGC specification begins in the early embryo as a result of a highly orchestrated combination of transcriptional and epigenetic mechanisms. Understanding the molecular events that lead to proper PGC development will facilitate the development of new treatments for human infertility as well as species conservation. This article describes the latest, most relevant findings about the mechanisms of PGC formation, emphasizing human PGC. It also discusses our own laboratory's progress in using transdifferentiation protocols to derive human PGCs (hPGCs). Our preliminary results arose from our pursuit of a sequential hPGC induction strategy that starts with the repression of lineage-specific factors in the somatic cell, followed by the reactivation of germ cell-related genes using specific master regulators, which can indeed reactivate germ cell-specific genes in somatic cells. While it is still premature to assume that fully functional human gametes can be obtained in a dish, our results, together with those recently published by others, provide strong evidence that generating their precursors, PGCs, is within reach.

Inhibin-B secretion and FSH isoform distribution may play an integral part of follicular selection in the natural menstrual cycle


The aim of the present paper is to expand the concept on how follicular selection takes place in the follicular phase of the natural menstrual cycle. It is suggested that inhibin-B exerts a more intimate role in this process than previously understood. Inhibin-B shows a peak in the circulation around cycle day 7, simultaneous with selection of the dominant follicle, whereas levels of estradiol and inhibin-A only start to increase a few days later suggesting that inhibin-B is mainly responsible for downregulating pituitary FSH release. New data now demonstrate that the circulatory peak of inhibin-B is reflected by peak production of inhibin-B, in contrast to inhibin-A, in the selected follicle with a diameter of 10–12 mm, where concentrations are one thousand times higher than in the circulation. This high inhibin-B concentration also exerts paracrine effects, stimulating theca cell androgen production in concert with LH. New data now suggest that in the corresponding granulosa cells androgens upregulate FSH receptor (FSHR) and LH receptor (LHR) mRNA expression, which in turn stimulate CYP19a mRNA expression providing the follicles which most effectively undertake these processes with the best chance of becoming selected. Inhibin-B production is stimulated by FSH and it appears that the acidic isoforms of FSH induce inhibin-B secretion most efficiently thereby, for the first time, placing the changing FSH isoform profile during the follicular phase in a physiological context. Collectively, it appears that inhibin-B is an integral part of follicular selection in the normal menstrual cycle, exerting both endocrine and paracrine effects and facilitating continued growth of the selected follicle.

Recombinant fetuin-B protein maintains high fertilization rate in cumulus cell-free mouse oocytes


Study Question

Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate?

Summary Answer

Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts.

What is Known Already

Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate.

Study Design, Size, Duration

We fertilized batches of 20–30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching.

Participants/Materials, Setting, Methods

Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm.

Main Results and the Role of Chance

In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P = 0.039). Fetuin-B at ≥0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P = 0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P = 0.02 at 1 hour, P = 0.01 at 5 hours of IVM).

Limitations, Reasons for Caution

Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined.

Wider Implications of the Findings

The astacin-type protease ovastacin triggers definitive ZP hardening by cleaving the zona pellucida protein 2. Animal sera are known to inhibit premature ZP hardening. The addition of rFetuB to the culture medium of oocytes could increase IVF rates by the inhibition of premature ZP hardening. In this regard, the results could be useful for clinical activity.

Large Scale Data


Study Funding/Competing Interest(s)

The research was supported by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University. The authors ED, JF and WJD are named inventors on a patent application of RWTH Aachen University covering the use of fetuin-B in ovary and oocyte culture.

Down-regulation of the liver-derived plasma protein fetuin-B mediates reversible female infertility


Study question Does antisense oligonucleotide (ASO)-mediated down-regulation of serum fetuin-B cause infertility like fetuin-B gene deficiency in female mice? Summary answer Pharmacological fetuin-B down-regulation by ASO therapy results in reversible infertility in female mice. What is known already Female fetuin-B deficient (Fetub–/–) mice are infertile owing to premature zona pellucida (ZP) hardening. Enzyme activity studies demonstrated that fetuin-B is a potent and highly specific inhibitor of the zona proteinase ovastacin, which cleaves ZP protein 2 (ZP2) and thus mediates definitive ZP hardening. Study design, size, duration Ten fetuin-B ASO boli (100 mg/kg) were injected s.c. over 20 days in 12 female mice, and 10 phosphate-buffered saline (PBS)-treated mice were used as control. At day 20 females were mated to evaluate fetuin-B as a potential molecular target for contraception. ASO and PBS treatment was continued for ten injections. After treatment cessation at day 50, mating was continued to investigate if infertility was reversible. Participants/materials, setting, methods We generated fetuin-B/ovastacin double deficient (Fetub–/–, Astl–/–) mice by conventional breeding to test if fertility of Fetub–/– female mice was restored when the target proteinase would likewise be deleted. At least five matings with each female genotype (Fetub–/– single deficient, Astl–/– single deficient, Fetub–/–, Astl–/– double deficient) were performed. To test the contraceptive effect of fetuin-B down-regulation, 22 female mice (6–13 weeks old) were treated with repetitive boli of 100 mg/kg fetuin-B ASO (n = 12) or PBS (n = 10) and mated continuously. Serum fetuin-B was determined by immunoblot before, during and after the ASO treatment. After 3 weeks of ASO treatment, in 6 females Fetub mRNA in liver was analyzed by PCR, and six PBS-treated females were used as control. Aspartate (AST) and alanine aminotransferase (ALT) were also measured in serum of six mice in each group. To determine the minimum permissive serum fetuin-B concentration required for successful fertilization IVF was performed in five fetuin-B ASO-treated mice. As a control, six females were injected with control oligonucleotides and six females were left untreated. Main results and the role of chance Fertility of Fetub–/– female mice was restored by additional ovastacin deficiency (Astl–/–). Unlike Fetub–/– mice, female Fetub–/–, Astl–/– mice were fertile, confirming ovastacin as a primary molecular target of fetuin-B. At day 20, after receiving 10 fetuin-B ASO boli, serum fetuin-B was down-regulated to 8 ± 6% (mean ± SD) of baseline level. Fetuin-B down-regulation was confirmed at the mRNA level. Fetuin-B ASO-treated females had 12.1 ± 3.1% of the liver Fetub mRNA level seen in PBS-treated females. In the following mating study, 11 out of 12 mated females failed to become pregnant during 50 days of ASO treatment and continuous mating from day 20 onwards. IVF of oocytes derived from ASO-treated females suggested that a serum fetuin-B level of less than 10 µg/ml was required to prevent pregnancy. Withdrawal of ASO treatment normalized serum fetuin-B and restored fertility; all female mice became pregnant and had litters within 60.3 ± 35.9 days after cessation of ASO treatment. The first litter was significantly smaller than that of control mice (4.6 ± 2.3 versus 6.7 ± 1.8 pups, n = 20, P = 0.04) but the smaller litter size was only temporary. The size of the second litter was similar to the first litter of control mice (7.6 ± 1.3 versus 6.7 ± 1.8 pups, n = 18, P = 0.25). Limitations, reasons for caution[...]

Normalization matters: tracking the best strategy for sperm miRNA quantification


Study Question What is the most reliable normalization strategy for sperm microRNA (miRNA) quantitative Reverse Transcription Polymerase Chain Reactions (qRT-PCR) using singleplex assays? Summary Answer The use of the average expression of hsa-miR-100-5p and hsa-miR-30a-5p as sperm miRNA qRT-PCR data normalizer is suggested as an optimal strategy. What is Known Already Mean-centering methods are the most reliable normalization strategies for miRNA high-throughput expression analyses. Nevertheless, specific trustworthy reference controls must be established in singleplex sperm miRNA qRT-PCRs. Study Design, Size Duration Cycle threshold (Ct) values from previously published sperm miRNA expression profiles were normalized using four approaches: (i) Mean-Centering Restricted (MCR) method (taken as the reference strategy); (ii) expression of the small nuclear RNA RNU6B; (iii) expression of four miRNAs selected by the Concordance Correlation Restricted (CCR) algorithm: hsa-miR-100-5p, hsa-miR-146b-5p, hsa-miR-92a-3p and hsa-miR-30a-5p; (iv) the combination of two of these miRNAs that achieved the highest proximity to MCR. Participants/Materials, Setting, Methods Expression profile data from 736 sperm miRNAs were taken from previously published studies performed in fertile donors (n = 10) and infertile patients (n = 38). For each tested normalizer molecule, expression ubiquity and uniformity across the different samples and populations were assessed as indispensable requirements for being considered as valid candidates. The reliability of the different normalizing strategies was compared to MCR based on the set of differentially expressed miRNAs (DE-miRNAs) detected between populations, the corresponding predicted targets and the associated enriched biological processes. Main Results and the Role of Chance All tested normalizers were found to be ubiquitous and non-differentially expressed between populations. RNU6B was the least uniformly expressed candidate across samples. Data normalization through RNU6B led to dramatically misguided results when compared to MCR outputs, with a null prediction of target genes and enriched biological processes. Hsa-miR-146b-5p and hsa-miR-92a-3p were more uniformly expressed than RNU6B, but their results still showed scant proximity to the reference method. The highest resemblance to MCR was achieved by hsa-miR-100-5p and hsa-miR-30a-5p. Normalization against the combination of both miRNAs reached the best proximity rank regarding the detected DE-miRNAs (Area Under the Curve = 0.8). This combination also exhibited the best performance in terms of the target genes predicted (72.3% of True Positives) and their corresponding enriched biological processes (70.4% of True Positives). Large Scale Data Not applicable. Limitations, Reasons for Caution This study is focused on sperm miRNA qRT-PCR analysis. The use of the selected normalizers in other cell types or tissues would still require confirmation. Wider Implications of the Findings The search for new fertility biomarkers based on sperm miRNA expression using high-throughput assays is one of the upcoming challenges in the field of reproductive genetics. In this context, validation of the results using singleplex assays would be mandatory. The normalizer strategy suggested in this study would provide a universal option in this area, allowing for normalization of the validated data without causing meaningful variations of the results. Instead, qRT-PCR data normalization by RNU6B should be discarded in sperm-miRNA expression studies. Study Funding/Competing Interest(S) This work was supported by the 2014/SGR00524 project (Agència de Gestió d'Ajuts Universitaris i de Recerca, Generalitat de Catalunya, Spain) and UAB CF-180034 grant (Universitat Autònoma de Barcelona). Celia Corral-Vazquez is a recipient of a Personal Investigador en Formació grant UAB/PIF2015 (Universitat Aut&og[...]

Antigen unmasking enhances visualization efficacy of the oocyte activation factor, phospholipase C zeta, in mammalian sperm


Study Question Is it possible to improve clinical visualization of phospholipase C zeta (PLC) as a diagnostic marker of sperm oocyte activation capacity and male fertility? Summary Answer Poor PLC visualization efficacy using current protocols may be due to steric or conformational occlusion of native PLC, hindering antibody access, and is significantly enhanced using antigen unmasking/retrieval (AUM) protocols. What is Known Already Mammalian oocyte activation is mediated via a series of intracellular calcium (Ca2+) oscillations induced by sperm-specific PLC. PLC represents not only a potential clinical therapeutic in cases of oocyte activation deficiency but also a diagnostic marker of sperm fertility. However, there are significant concerns surrounding PLC antibody specificity and detection protocols. Study Design, Size Duration Two PLC polyclonal antibodies, with confirmed PLC specificity, were employed in mouse, porcine and human sperm. Experiments evaluated PLC visualization efficacy, and whether AUM improved this. Antibodies against two sperm-specific proteins [post-acrosomal WW-binding protein (PAWP) and acrosin] were used as controls. Participants/Materials, Setting, Methods Aldehyde- and methanol-fixed sperm were subject to immunofluorescence analysis following HCl exposure (pH = 0.1–0.5), acid Tyrode's solution exposure (pH = 2.5) or heating in 10 mM sodium citrate solution (pH = 6.0). Fluorescence intensity of at least 300 cells was recorded for each treatment, with three independent repeats. Main Results and the Role of Chance Despite high specificity for native PLC following immunoblotting using epitope-specific polyclonal PLC antibodies in mouse, porcine and human sperm, immunofluorescent visualization efficacy was poor. In contrast, sperm markers PAWP and acrosin exhibited relatively impressive results. All methods of AUM on aldehyde-fixed sperm enhanced visualization efficacy for PLC compared to visualization efficacy before AUM (P < 0.05 for all AUM interventions), but exerted no significant change upon PAWP or acrosin immunofluorescence following AUM. All methods of AUM enhanced PLC visualization efficacy in mouse and human methanol-fixed sperm compared to without AUM (P < 0.05 for all AUM interventions), while no significant change was observed in methanol-fixed porcine sperm before and after. In the absence of aldehyde-induced cross-linkages, such results suggest that poor PLC visualization efficacy may be due to steric or conformational occlusion of native PLC, hindering antibody access. Importantly, examination of sperm from individual donors revealed that AUM differentially affects observable PLC fluorescence, and the proportion of sperm exhibiting detectable PLC fluorescence in sperm from different males. Limitations, Reasons for Caution Direct correlation of fertility outcomes with the level of PLC in the sperm samples studied was not available. Such analyses would be required in future to determine whether the improved methodology for PLC visualization we propose would indeed reflect fertility status. Wider Implications of the Findings We propose that AUM alters conformational interactions to enhance PLC epitope availability and visualization efficacy, supporting prospective application of AUM to reduce misinterpretation in clinical diagnosis of PLC-linked male infertility. Our current results suggest that it is perhaps prudent that previous studies investigating links between PLC and fertility parameters are re-examined in the context of AUM, and may pave the way for future work to answer significant questions such as how PLC appears to be kept in an inactive form in the sperm. Large Scale Data Not applicable. Study Funding/Competing Interest(S) J.K. is supported by a Health Fellowship award from the National Institute for Social Care and Health Research (NISCHR). M.N. is supported by a Marie Curie Intra-Europe[...]