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Corina Isabel Schanzenbach, Sandra Milena Bernal-Ulloa, Vera Anna van der Weijden, Michael W. Pfaffl, Mathias Büttner, Annegret Wünsch and Susanne E Ulbrich

Preimplantation bovine blastocyst supernatants exhibit sex-dependent antiviral activity, due to the ruminant pregnancy recognition signal Interferon tau (IFNT). Differing potencies of IFNT variants have been supposed as cause, although evidence remains scarce. Here, we aimed at quantifying the sex-dependent IFNT production on transcriptional, translational, and biological activity level in bovine blastocysts, to elucidate the origin of differences in antiviral activity between male and female blastocysts. Day 8 bovine blastocysts were co-cultured with endometrial stroma cells for 48 hours. The embryonic IFNT mRNA expression was determined by quantitative reverse transcription followed by polymerase chain reaction (RT-qPCR). Additionally, the IFNT protein concentration was determined using a sensitive in-house developed IFNT-specific Enzyme-linked-immunosorbent Assay (ELISA). The biological activity was assessed by quantifying the response of Interferon stimulated gene (ISG) expression in endometrial stroma cells. While the IFNT specific ELISA displayed a limit of detection of 7.3 pg/mL, the stroma cell culture system showed to react to as little as 0.1 pg/mL IFNT in RT-qPCR analysis. The female blastocysts had a significant, 5.6-fold, 3.6-fold, and 5.2-fold higher IFNT production than male blastocysts as determined by transcript abundance, protein concentration and, protein activity, respectively. Additionally, all parameters correlated positively, and therefore, we conclude that female blastocysts most likely have an increased IFNT gene and protein expression rather than expressing more potent IFNT variants.

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Jessica Elizabeth Maree Dunleavy, Moira O'Bryan, Peter G. Stanton and Liza O'Donnell

As germ cells progress through spermatogenesis, they undergo a dramatic transformation, wherein a single, diploid spermatogonial stem cell ultimately produces thousands of highly specialised, haploid spermatozoa. The cytoskeleton is an integral aspect of all eukaryotic cells. It concomitantly provides both structural support and functional pliability, performing key roles in many fundamental processes including, motility, intracellular trafficking, differentiation and cell division. Accordingly, cytoskeletal dynamics underlie many key spermatogenic processes. This review summarises the organisational and functional aspects of the four major cytoskeletal components (actin, microtubules, intermediate filaments and septins) during the various spermatogenic phases in mammals. We focus on the cytoskeletal machinery of both germ cells and Sertoli cells, and thus highlight the critical importance of a dynamic and precisely regulated cytoskeleton for male fertility.

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Chizuru Ito, Kenji Yamatoya, Keiichi Yoshida, Lisa Fujimura, Hajime Sugiyama, Akiko Suganami, Yutaka Tamura, Masahiko Hatano, Kenji Miyado and Kiyotaka Toshimori

A number of sperm proteins are involved in the processes from gamete adhesion to fusion, but the underlying mechanism is still unclear. Here, we established a mouse mutant, the EQUATORIN-knockout (EQTN-KO, Eqtn / ) mouse model and found that the EQTN-KO males have reduced fertility and sperm–egg adhesion, while the EQTN-KO females are fertile. Eqtn / sperm were normal in morphology and motility. Eqtn / -Tg (Acr-Egfp) sperm, which were produced as the acrosome reporter by crossing Eqtn / with Eqtn +/+-Tg(Acr-Egfp) mice, traveled to the oviduct ampulla and penetrated the egg zona pellucida of WT females. However, Eqtn / males mated with WT females showed significant reduction in both fertility and the number of sperm attached to the zona-free oocyte. Sperm IZUMO1 and egg CD9 behaved normally in Eqtn / sperm when they were fertilized with WT egg. Another acrosomal protein, SPESP1, behaved aberrantly in Eqtn / sperm during the acrosome reaction. The fertility impairment of EQTN/SPESP1-double KO males lacking Eqtn and Spesp1 (Eqtn/Spesp1 / ) was more severe compared with that of Eqtn / males. Eqtn / -Tg (Eqtn) males, which were generated to rescue Eqtn / males, restored the reduced fertility.

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Silvia Sposini and Aylin C Hanyaloglu

Our understanding of G protein-coupled receptor (GPCR) signalling has significantly evolved over the past decade, whereby signalling not only occurs from the plasma membrane but continues, or is reactivated, following internalisation in to endosomal compartments. The spatial organisation of GPCRs is thus essential to decode dynamic and complex signals and to activate specific downstream pathways that elicit the appropriate cellular response. For the gonadotrophin hormone receptors, membrane trafficking has been demonstrated to play a significant role in regulating its signal activity that in turn would impact at physiological and even pathophysiological level. Here, we will describe the developments in our understanding of the role of ‘location’ in gonadotrophin hormone receptor signalling, and how these receptors have unveiled fundamental mechanisms of signal regulation likely to be pertinent for other GPCRs. We will also discuss the potential impact of spatially controlled gonadotrophin hormone receptor signalling in both health and disease, and the therapeutic possibilities this new understanding of these receptors, so key in reproduction, offers.

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Pacharawan Deenarn, Punsa Tobwor, Rungnapa Leelatanawit, Somjai Wongtriphop, Jutatip Khudet, Nitsara Karoonuthaisiri, Vanicha Vichai and Wananit Wimuttisuk

The delay in ovarian maturation in farmed black tiger shrimp Penaeus monodon has resulted in the widespread practice of feeding broodstock with the polychaete Perinereis nuntia and their unilateral eyestalk ablation. Although this practice alters fatty acid content in shrimp ovaries and hepatopancreas, its effects on fatty acid regulatory genes are yet to be systematically examined. Here, microarray analysis was performed on hepatopancreas and ovary cDNA collected from P. monodon at different ovarian maturation stages, revealing that 72 and 58 genes in fatty acid regulatory pathways were differentially expressed in hepatopancreas and ovaries respectively. Quantitative real-time PCR analysis revealed that ovarian maturation was associated with higher expression levels of acetyl-CoA acetyltransferase, acyl-CoA dehydrogenase, acyl-CoA oxidase 3 and long-chain fatty acid transport protein 4 in hepatopancreas, whereas the expression levels of 15 fatty acid regulatory genes were increased in shrimp ovaries. To distinguish the effects of different treatments, transcriptional changes were examined in P. monodon with stage 1 ovaries before polychaete feeding, after 1 month of polychaete feeding and after eyestalk ablation. Polychaete feeding resulted in lower expression levels of enoyl-CoA hydratase and acyl-CoA synthetase medium-chain family member 4, while the expression level of phosphatidylinositide phosphatase SAC1 was higher in shrimp hepatopancreas and ovaries. Additionally, eyestalk ablation resulted in a higher expression level of long-chain fatty acid-CoA ligase 4 in both tissues. Together, our findings describe the dynamics of fatty acid regulatory pathways during crustacean ovarian development and provide potential target genes for alternatives to eyestalk ablation in the future.

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Jun Yin, Bing Ni, Yi-dong Yang, Zhong-wei Tang, Zhi-qi Gao, Lan Feng, Wei-gong Liao and Yu-qi Gao

Autophagy and apoptosis are interlocked in an extensive crosstalk. Our previous study demonstrated that hypotonic hypoxia-induced marked apoptosis of a spermatocyte-derived cell line (GC-2). However, whether hypoxia-induced apoptosis is mediated by inhibition of autophagy under hypoxic conditions remains unclear. In this study, GC-2 cells were cultured in 1% O2 and harvested at different time points. Autophagy was determined by acridine orange staining, cyto-ID staining, mCherry-GFP-LC3B adenovirus transfection and Western blotting for various autophagy markers. Apoptosis was detected by TUNEL staining, flow cytometry, JC-1 staining and Western blotting of apoptosis-related proteins. We found that hypoxia-induced apoptosis of GC-2 cells through mitochondrial and death receptor pathways and inhibited autophagic flux in GC-2 cells in a time-dependent manner. However, while marked autolysosome formation was observed in GC-2 cells before 24-h culture in hypoxic conditions, apparent apoptosis was observed only after 24-h culture in hypoxic conditions. Caspase-8 siRNA treatment induced cell survival, accompanied by induction of the mature autophagosome, acidic vesicular organelle formation and autophagic flux. Furthermore, Beclin-1 overexpression markedly attenuated the impairment of spermatogenesis in mice by inhibiting apoptosis of spermatocytes. The results of this study demonstrate that hypoxia inhibits autophagy, which further enhances hypoxia-induced apoptosis of mouse spermatocytes by promoting caspase-8 activation in a time-dependent manner, suggesting that combined application of apoptosis inhibition and autophagy activation might be a therapeutic strategy for treating hypoxia-induced male infertility.

Open access

Beverly G Reed, Samir N Babayev, Lucy X Chen, Bruce R Carr, R Ann Word and Patricia T Jimenez

MicroRNAs (miRs) are small molecules important for regulation of transcription and translation. The objective was to identify hormonally regulated miRs in human endometrial stromal cells and to determine the impact of the endocrine disruptor, bisphenol A (BPA), on those miRs. miR microarray analysis and multiple confirmatory cell preparations treated with 17β-estradiol (E2) and BPA altered miR-27b, let-7c, let-7e and miR-181b. Further, decidualization downregulated miR-27b. VEGFB and VEGFC were validated as targets of miR-27b. Identification of miR-27b target genes suggests that BPA and E2 downregulate miR-27b thereby leading to upregulation of genes important for vascularization and angiogenesis of the endometrium during the menstrual cycle and decidualization.

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Melissa L McCallum, Cindy A Pru, Andrea R Smith, Nicole C Kelp, Marc Foretz, Benoit Viollet, Min Du and James K Pru

Adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved heterotrimeric complex that acts as an intracellular energy sensor. Based on recent observations of AMPK expression in all structures of the female reproductive system, we hypothesized that AMPK is functionally required for maintaining fertility in the female. This hypothesis was tested by conditionally ablating the two catalytic alpha subunits of AMPK, Prkaa1 and Prkaa2, using Pgr-cre mice. After confirming the presence of PRKAA1, PRKAA2 and the active phospho-PRKAA1/2 in the gravid uterus by immunohistochemistry, control (Prkaa1/2 fl/fl) and double conditional knockout mice (Prkaa1/2 d/d) were placed into a six-month breeding trial. While the first litter size was comparable between Prkaa1/2 fl/fl and Prkaa1/2 d/d female mice (P = 0.8619), the size of all subsequent litters was dramatically reduced in Prkaa1/2 d/d female mice (P = 0.0015). All Prkaa1 /2 d/d female mice experienced premature reproductive senescence or dystocia by the fourth parity. This phenotype manifested despite no difference in estrous cycle length, ovarian histology in young and old nulliparous or multiparous animals, mid-gestation serum progesterone levels or uterine expression of Esr1 or Pgr between Prkaa1/2 fl/fl and Prkaa1/2 d/d female mice suggesting that the hypothalamic–pituitary–ovary axis remained unaffected by PRKAA1/2 deficiency. However, an evaluation of uterine histology from multiparous animals identified extensive endometrial fibrosis and disorganized stromal-glandular architecture indicative of endometritis, a condition that causes subfertility or infertility in most mammals. Interestingly, Prkaa1/2 d/d female mice failed to undergo artificial decidualization. Collectively, these findings suggest that AMPK plays an essential role in endometrial regeneration following parturition and tissue remodeling that accompanies decidualization.

Open access

Caroline M Allen, Federica Lopes, Rod T Mitchell and Norah Spears

Chemotherapy treatment is a mainstay of anticancer regimens, significantly contributing to the recent increase in childhood cancer survival rates. Conventional cancer therapy targets not only malignant but also healthy cells resulting in side effects including infertility. For prepubertal boys, there are currently no fertility preservation strategies in use, although several potential methods are under investigation. Most of the current knowledge in relation to prepubertal gonadotoxicity has been deduced from adult studies; however, the prepubertal testis is relatively quiescent in comparison to the adult. This review provides an overview of research to date in humans and animals describing chemotherapy-induced prepubertal gonadotoxicity, focusing on direct gonadal damage. Testicular damage is dependent upon the agent, dosage, administration schedule and age/pubertal status at time of treatment. The chemotherapy agents investigated so far target the germ cell population activating apoptotic pathways and may also impair Sertoli cell function. Due to use of combined chemotherapy agents for patients, the impact of individual drugs is hard to define, however, use of in vivo and in vitro animal models can overcome this problem. Furthering our understanding of how chemotherapy agents target the prepubertal testis will provide clarity to patients on the gonadotoxicity of different drugs and aid in the development of cytoprotective agents.

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Qianrong Qi, Yifan Yang, Kailin Wu and Qingzhen Xie

Recent studies revealed that TMEM16A is involved in several reproductive processes, including ovarian estrogen secretion and ovulation, sperm motility and acrosome reaction, fertilization and myometrium contraction. However, little is known about the expression and function of TMEM16A in embryo implantation and decidualization. In this study, we focused on the expression and regulation of TMEM16A in mouse uterus during early pregnancy. We found that TMEM16A is upregulated in uterine endometrium in response to embryo implantation and decidualization. Progesterone treatment could induce TMEM16A expression in endometrial stromal cells through progesterone receptor/c-Myc pathway, which is blocked by progesterone receptor antagonist or the inhibitor of c-Myc signaling pathway. Inhibition of TMEM16A by small molecule inhibitor (T16Ainh-A01) resulted in impaired embryo implantation and decidualization in mice. Treatment with either specific siRNA of Tmem16a or T16Ainh-A01 inhibited the decidualization and proliferation of mouse endometrial stromal cells. In conclusion, our results revealed that TMEM16A is involved in embryo implantation and decidualization in mice, compromised function of TMEM16A may lead to impaired embryo implantation and decidualization.