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Ruizhi Deng, Chengquan Han, Lu Zhao, Qing Zhang, Beifen Yan, Rui Cheng, Biao Wei, Peng Meng, Tingchao Mao, Yong Zhang and Jun Liu

Endogenous retroviruses (ERVs), which are abundant in mammalian genomes, can modulate the expression of nearby genes, and their expression is dynamic and stage-specific during early embryonic development in mice and humans. However, the functions and mechanisms of ERV elements in regulating embryonic development remain unclear. Here, we utilized several methods to determine the contribution of ERVs to the makeup and regulation of transcripts during embryonic genome activation (EGA). We constructed an ERV library and embryo RNA-seq library (IVF_2c and IVF_8c) of goat to serve as our research basis. The GO and KEGG analysis of nearby ERV genes revealed that some ERV elements may be associated with embryonic development. RNA-seq results were consistent with the features of EGA. To obtain the transcripts derived from the ERV sequences, we blasted the ERV sequences with embryonic transcripts and identified three lncRNAs and one mRNA that were highly expressed in IVF-8c rather than in IVF-2c (q-value <0.05). Then, we validated the expression patterns of nine ERV-related transcripts during early developmental stages and knocked down three high-expression transcripts in EGA. The knockdown of lncRNA TCONS_00460156 or mRNA HSD17B11 significantly decreased the developmental rate of IVF embryos. Our findings suggested that some transcripts from ERVs are essential for the early embryonic development of goat, and analyzing the ERV expression profile during goat EGA may help elucidate the molecular mechanisms of ERV in regulating embryonic development.

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D Ioannou, J Fortun and H G Tempest

Infertility is relatively common affecting approximately 1-in-6 couples. Although the genetic basis of infertility is increasingly being uncovered, the contribution of male infertility often remains unexplained. The leading cause of pregnancy loss and cognitive impairment in humans is chromosome aneuploidy. Sperm aneuploidy is routinely evaluated by fluorescence in situ hybridization. The majority of studies have reported similar findings, namely: (1) all men produce aneuploid sperm; (2) certain chromosomes are more prone to undergo chromosome nondisjunction; (3) infertile men typically have significantly higher levels of sperm aneuploidy compared to controls and (4) the level of aneuploidy is often correlated with the severity of the infertility. Despite this, sperm aneuploidy screening is rarely evaluated in the infertility clinic. Within recent years, there appears to be renewed interest in the clinical relevance of sperm aneuploidy. We shall examine the gender differences in meiosis between the sexes and explore why less emphasis is placed on the paternal contribution to aneuploidy. Increased sperm aneuploidy is often perceived to be modest and not clinically relevant, compared to the female contribution. However, even small increases in sperm aneuploidy may impact fertility and IVF cycle outcomes. Evidence demonstrating the clinical relevance of sperm aneuploidy will be discussed, as well as some of the challenges precluding widespread clinical implementation. Technological developments that may lead to widespread clinical implementation will be discussed. Recommendations will be suggested for specific patient groups that may benefit from sperm aneuploidy screening and whether preimplantation genetic testing for aneuploidy should be discussed with these patients.

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Nicolas J Fasel, Kevin McMillian, Ulrike Jakop, Laurent Méné-Saffrané, Kathrin M Engel, Michel Genoud, Karin Müller and Philippe Christe

Biochemical properties of polyunsaturated fatty acids (PUFAs) are fundamental to sperm movements. Amongst all adjustments operated during epididymal maturation, sperm membrane lipid composition is remodelled. Specifically, the proportion of PUFAs usually increases from the caput towards the cauda epididymidis. In mammals, PUFAs are predominantly acquired through the diet, which can consequently impact male fertility. We aimed at analysing to what extent n-6 and n-3 PUFAs are incorporated into sperm in the Seba’s short-tailed bat (Carollia perspicillata), and at demonstrating the effect of the sperm fatty acid composition on sperm mobility. We therefore provided food varying in fatty acid composition to males of C. perspicillata and measured the fatty acid composition and mobility traits in spermatozoa collected from the caput and cauda epididymides. We found that n-6 and n-3 PUFAs and saturated fatty acids were significantly related to sperm velocity but not to the proportion of progressive sperm (i.e. motility). Concomitant to an increase in sperm velocity, the level of fatty acid saturation increased from the caput to the cauda epididymidis, while the proportion of PUFAs remained similar along the epididymis. A reduction in n-6 PUFAs counterbalanced an increase in n-3 PUFAs. The food treatments did not affect the sperm fatty acid composition. Our results suggest that a precise endogenous control rather than dietary effects determines sperm fatty acid composition in C. perspicillata.

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Vijay Pratap Singh, Wei-ting Yueh, Jennifer L. Gerton and Francesca E. Duncan

Eighteen histone deacetylases exist in mammals. The class 1 histone deacetylases HDAC1 and HDAC2 are important for oogenesis and fertility in mice, likely via their effects on histones. The reproductive function of HDAC8, another class 1 enzyme, has not been explored. One key target of HDAC8 is the SMC3 subunit of cohesin, an essential complex mediating sister chromatid cohesion and chromosome segregation. In current models, HDAC8 activity is required for SMC3 recycling, but this function should be dispensable in oocytes since cohesion is established during pre-meiotic S phase and maintained until meiotic resumption during ovulation. Whether other oocyte specific HDAC8 mediated deacetylation events are required for oogenesis and female fertility is unknown. We used two cre drivers to remove Hdac8 at specific stages of oocyte development to address whether HDAC8 is required for female fertility in mice. When HDAC8 was knocked out in oocytes in primary and later stage follicles (Zp3-Cre), oogenesis and folliculogenesis appeared normal and mice were fertile. However, females were sub-fertile when HDAC8 was knocked out prior to pre-meiotic S phase and cohesion establishment (Vasa-Cre). This subfertility was independent of chromosome segregation errors during meiosis but rather appeared to be the result of defects in oogenesis that resulted in smaller fully grown oocytes with a reduced ability to resume meiosis. In all cases, we did not observe compensatory changes in HDAC1, HDAC2, and HDAC3 levels. Thus, although oocyte-specific expression of HDAC8 is not essential for mouse oogenesis after meiotic S phase, it contributes to optimal fertility.

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E Wolff, M M Suplicki and R Behr

Primordial germ cells (PGCs) are the embryonic precursors of spermatozoa and eggs. In mammals, PGCs arise early in embryonic development and migrate from their tissue of specification over a significant distance to reach their destinations, the genital ridges. However, the exact mechanism of translocation is still debated. A study on human embryos demonstrated a very close spatial association between migrating PGCs and developing peripheral nerves. Thus, it was proposed that peripheral nerves act as guiding structures for migrating PGCs. The goal of the present study is to test whether the association between nerves and PGCs may be a human-specific finding or whether this represents a general strategy to guide PGCs in mammals. Therefore, we investigated embryos of different developmental stages from the mouse and a non-human primate, the marmoset monkey (Callithrix jacchus), covering the phase from PGC emergence to their arrival in the gonadal ridge. Embryo sections were immunohistochemically co-stained for tubulin beta-3 chain (TUBB3) to visualise neurons and Octamer-binding protein 4 (OCT4 (POU5F1)) as marker for PGCs. The distance between PGCs and the nearest detectable neuron was measured. We discovered that in all embryos analysed of both species, the majority of PGCs (>94%) was found at a minimum distance of 50 µm to the closest neuron and, more importantly, that the PGCs had reached the gonads before any TUBB3 signal could be detected in the vicinity of the gonads. In conclusion, our data indicate that PGC migration along peripheral nerves is not a general mechanism in mammals.

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Parag Parekh, Thomas Xavier Garcia and Marie-claude Hofmann

Sertoli cells regulate male germ cell proliferation and differentiation and are a critical component of the spermatogonial stem cell (SSC) niche, where homeostasis is maintained by the interplay of several signaling pathways and growth factors. These factors are secreted by Sertoli cells located within the seminiferous epithelium, and by interstitial cells residing between the seminiferous tubules. Sertoli cells and peritubular myoid cells produce glial cell line-derived neurotrophic factor (GDNF), which binds to the RET/GFRA1 receptor complex at the surface of undifferentiated spermatogonia. GDNF is known for its ability to drive SSC self-renewal and proliferation of their direct cell progeny. Even though the effects of GDNF are well studied, our understanding of the regulation its expression is still limited. The purpose of this review is to discuss how GDNF expression in Sertoli cells is modulated within the niche, and how these mechanisms impact germ cell homeostasis.

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Valeria Merico, Juan Pablo Luaces, Luis Francisco Rossi, Paola Rebuzzini, Maria Susana Merani, Maurizio Zuccotti and Silvia Garagna

In nature, mammalian seasonal breeders undergo spermatogenetic arrest during the non-breeding season. In the large hairy armadillo Chaetophractus villosus, testis regression initiates with immature post-meiotic germ cells sloughing into the tubule lumen and continues with the death of the remaining spermatocytes. At the end of the regression period, only spermatogonia and Sertoli cells persist in the seminiferous epithelium. It has been suggested that cell sloughing is determined by changes in the adhesion complexes between Sertoli cells and spermatids, which are mediated by low intra-testicular testosterone levels. By immunofluorescence and Western blotting we studied key proteins of the N-cadherin/N-cadherin and A6B1-integrin/laminin interlocks that contribute to the complex Sertoli/spermatid adhesion system throughout the eight stages of the seminiferous epithelium cycle in the comparison between active and regressing testes. In active testis, B1-integrin, laminin G3, N-cadherin, B-catenin, P-B-catenin-Tyr654, FAK, P-FAK-Tyr397, SRC, P-SRC-Tyr416 proteins present a spermatogenetic cycle-dependent localisation pattern, unmaintained in regressing testes. In the latter, quantitative variations and changes in the phosphorylation state of protein FAK, SRC and B-catenin contribute to the disassembly of the N-cadherin/N-cadherin and A6B1-integrin/laminin interlocks, thus promoting the massive release of immature spermatids.

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Michal Zigo, Vera Jonakova, Pavla Manaskova-Postlerova, Karl Kerns and Peter Sutovsky

We studied the participation of the ubiquitin proteasome system (UPS) in spermadhesin release during in vitro capacitation (IVC) of domestic boar spermatozoa. At ejaculation, boar spermatozoa acquire low molecular weight (8-16 kDa) seminal plasma proteins, predominantly spermadhesins, aggregated on the sperm surface. Due to their arrangement, such aggregates are relatively inaccessible to antibody labeling. As a result of de-aggregation and release of the outer layers of spermadhesins from the sperm surface during IVC, antibody labeling becomes feasible in the capacitated spermatozoa. In vivo, the capacitation-induced shedding of spermadhesins from the sperm surface is associated with the release of spermatozoa from the oviductal sperm reservoir. We took advantage of this property to perform image-based flow cytometry to study de-aggregation and shedding of boar spermadhesins (AQN, AWN, PSP protein families) and boar DQH (BSP1) sperm surface protein which induces higher fluorescent intensity in capacitated vs. ejaculated spermatozoa. Addition of a proteasomal inhibitor (100 µM MG132) during IVC significantly reduced fluorescence intensity of all studied proteins (P<0.05) compared to vehicle control IVC. Western blot detection of spermadhesins did not support their retention during IVC with proteasomal inhibition (P>0.99) but showed the accumulation of DQH (P=0.03) during IVC, compared to vehicle control IVC. Our results thus demonstrate that UPS participates in the de-aggregation of spermadhesins and DQH protein from the sperm surface during capacitation, with a possible involvement in sperm detachment from the oviductal sperm reservoir and/or sperm-zona pellucida interactions.

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Yumiko Miyazaki, Akihito Horie, Hirohiko Tani, Masashi Ueda, Asuka Okunomiya, Koh Suginami, Eiji Kondoh, Tsukasa Baba, Ikuo Konishi, Tamayuki Shinomura and Yukiyasu Sato

The endometrium extracellular matrix (ECM) is essential for embryo implantation. Versican, a large chondroitin sulfate proteoglycan that binds hyaluronan and forms large ECM aggregates, can influence fundamental physiological phenomena, such as cell proliferation, adhesion and migration. The present study investigated the possible role of versican in human embryo implantation. Versican V1 expression and secretion in human endometrial epithelial cells (EECs) was most prominent in the mid-secretory phase. Versican expression in EECs significantly increased after treatment with estrogen and progesterone, but not by estrogen alone. We also established versican V1-overexpressing Ishikawa (endometrial cancer cell line) cells (ISKW-V1), versican V3-overexpressing (ISKW-V3) and control GFP-overexpressing (ISKW-GFP) Ishikawa cells. By the in vitro implantation model, the attachment ratio of BeWo (choriocarcinoma cell line) spheroids to the monolayer of ISKW-V1, but not of ISKW-V3, was found significantly enhanced compared with attachment to the ISKW-GFP monolayer. The conditioned medium derived from ISKW-V1 (V1-CM) also promoted the attachment of BeWo spheroids to the ISKW monolayer. However, this attachment-promoting effect was abolished when V1-CM was pretreated with chondroitinase ABC, which degrades chondroitin sulfate. Therefore, out of the ECM components, versican V1 may facilitate human embryo implantation.

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Mariana Regueira, Agostina Gorga, Gustavo Marcelo Rindone, Eliana Herminia Pellizzari, Selva Beatriz Cigorraga, María Noel Galardo, María Fernanda Riera and Silvina Beatriz Meroni

The presence of lipid droplets (LD) and the utilization of fatty acids (FA) as a source of energy are Sertoli cell (SC) putative characteristics. It is well known that SCs can phagocyte and degrade apoptotic germ cells (AGC) resulting in increasing lipid content and ATP levels. A relationship between the regulation of lipid storage and of lipid oxidation in SC might be envisaged. The aim of this study was to analyze whether AGC and FA are able to simultaneously regulate molecular mechanisms involved in lipid storage and in FA oxidation in SC. The experimental model utilized in this study consisted in SC cultures obtained from 20-day-old rats that were co-cultured with AGC or treated with palmitic acid (PA, 500 μM) for 24 and 48 h. AGC and PA increase LD, triacylglycerol (TAG) content and mRNA levels of Plin1, Plin2, Plin3 (proteins involved in TAG storage). Simultaneously, AGC and PA rise the extent of FA oxidation and mRNA levels of Cpt1 and Lcad (proteins involved in FA degradation). Results also show that peroxisome proliferator-activated receptor (PPAR) transcriptional activity, transcription factor which participate in lipid metabolism regulation, increases by AGC and PA treatment in SC. Additionally, the presence of a PPARg antagonist decreases the upregulation of LD content and Plin1 expression. Similarly, the presence of a PPARb/d antagonist reduces the increase in FA oxidation and Cpt1 mRNA levels. Altogether these results suggest that AGC and FA, which probably generate PPAR ligands, regulate lipid storage and fatty acid utilization, contributing to the energy homeostasis in the seminiferous tubules.