Through the smoke: Use of in vivo and in vitro cigarette smoking models to elucidate its effect on female fertility

A finite number of oocytes are established within the mammalian ovary prior to birth to form a precious ovarian reserve. Damage to this limited pool of gametes by environmental factors such as cigarette smoke and its constituents therefore represents a significant risk to a woman's reproductive capacity. Although evidence from human studies to date implicates a detrimental effect of cigarette smoking on female fertility, these retrospective studies are limited and present conflicting results. In an effort to more clearly understand the effect of cigarette smoke, and its chemical constituents, on female fertility, a variety of in vivo and in vitro animal models have been developed. This article represents a systematic review of the literature regarding four of experimental model types: 1) direct exposure of ovarian cells and follicles to smoking constituents' in vitro, 2) direct exposure of whole ovarian tissue with smoking constituents in vitro, 3) whole body exposure of animals to smoking constituents and 4) whole body exposure of animals to cigarette smoke. We summarise key findings and highlight the strengths and weaknesses of each model system, and link these to the molecular mechanisms identified in smoke-induced fertility changes

The use of C57Bl/6 × CBA F1 hybrid cross as a model for human age-related oocyte aneuploidy

Oocyte numbers decrease whereas the incidence of aneuploidy increases as women age. The molecular mechanisms underpinning this age-related decline in oocyte quality are not completely understood. Human oocytes are particularly error prone, with reports of aneuploidy rates as high as 50-60% (Fragouli et al., 2011; Kuliev et al., 2011). Mouse oocytes, in contrast, are generally more resilient to age-related aneuploidy, with different stains harboring disparate susceptibilities to chromosome segregation errors. This is clearly observed with aneuploidy rates as low as 9% (C57Bl/6 mice, 17-19 months) to 25% (B6D2F1/J mice, 16-19 months), but may be as high as 33% (MF1 mice, 15-17 months) to 43% (CD1 mice, 19-25 months) (Chiang et al., 2010; Sebestova et al., 2012; Shomper et al., 2014; Yun et al., 2014). Such variability in murine aneuploidy rates has hampered investigations into the causes of aged-related aneuploidy, with even the highest aneuploidy rates failing to reach those presented in older women

Motoring through : the role of kinesin superfamily proteins in female meiosis

BACKGROUND: The kinesin motor protein family consists of 14 distinct subclasses and 45 kinesin proteins in humans. A large number of these proteins, or their orthologues, have been shown to possess essential function(s) in both the mitotic and the meiotic cell cycle. Kinesins have important roles in chromosome separation, microtubule dynamics, spindle formation, cytokinesis and cell cycle progression. This article contains a review of the literature with respect to the role of kinesin motor proteins in female meiosis in model species. Throughout, we discuss the function of each class of kinesin proteins during oocyte meiosis, and where such data are not available their role in mitosis is considered. Finally, the review highlights the potential clinical importance of this family of proteins for human oocyte quality. OBJECTIVE AND RATIONALE: To examine the role of kinesin motor proteins in oocyte meiosis. SEARCH METHODS: A search was performed on the Pubmed database for journal articles published between January 1970 and February 2017. Search terms included 'oocyte kinesin' and 'meiosis kinesin' in addition to individual kinesin names with the terms oocyte or meiosis. OUTCOMES: Within human cells 45 kinesin motor proteins have been discovered, with the role of only 13 of these proteins, or their orthologues, investigated in female meiosis. Furthermore, of these kinesins only half have been examined in mammalian oocytes, despite alterations occurring in gene transcripts or protein expression with maternal ageing, cryopreservation or behavioral conditions, such as binge drinking, for many of them. WIDER IMPLICATIONS: Kinesin motor proteins have distinct and important roles throughout oocyte meiosis in many non-mammalian model species. However, the functions these proteins have in mammalian meiosis, particularly in humans, are less clear owing to lack of research. This review brings to light the need for more experimental investigation of kinesin motor proteins, particularly those associated with maternal ageing, cryopreservation or exposure to environmental toxicants

Germ cell specific overactivation of WNT/βcatenin signalling has no effect on folliculogenesis but causes fertility defects due to abnormal foetal development

All the major components of the WNT signalling pathway are expressed in female germ cells and embryos. However, their functional relevance in oocyte biology is currently unclear. We examined ovaries collected from TCFGFP mice, a well-known Wnt reporter mouse model, and found dynamic changes in the Wnt/βcatenin signalling activity during different stages of oocyte development and maturation. To understand the functional importance of Wnt signalling in oocytes, we developed a mouse model with the germ cell-specific constitutive activation of βcatenin using cre recombinase driven by the DEAD (Asp-Glu-Ala-Asp) box protein 4 (Ddx4) gene promoter. Histopathological and functional analysis of ovaries from these mutant mice (Ctnnb1ex3cko) showed no defects in ovarian functions, oocytes, ovulation and early embryonic development. However, breeding of the Ctnnb1ex3cko female mice with males of known fertility never resulted in birth of mutant pups. Examination of uteri from time pregnant mutant females revealed defects in ectoderm differentiation leading to abnormal foetal development and premature death. Collectively, our work has established the role of active WNT/βcatenin signalling in oocyte biology and foetal development, and provides novel insights into the possible mechanisms of complications in human pregnancy such as repeated spontaneous abortion, sudden intrauterine unexpected foetal death syndrome and stillbirth

Kif4 Is Essential for Mouse Oocyte Meiosis

<div><p>Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age.</p></div

Kif4 co-localizes and interacts with outer kinetochore Ndc80 throughout meiosis.

<p>(A) Fluorescent immunolocalization of Kif4 and Ndc80 at GV, MI and MII. MI oocytes are counter labelled with the inner kinetochore marker ACA (red). Secondary only controls reveal nonspecific antibody binding to the zona pellucida only. (B) Positive proximately ligation assay (red) between Kif4 and Ndc80 at GV, MI and MII is indicative of protein-protein interaction. Insets highlight interaction on chromosomes (See Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170650#pone.0170650.s001" target="_blank">S1D Fig</a> for additional examples). PLA negative control of Kif4 with testis specific Piwil1 antibodies reveal no nonspecific signal amplification. DNA is counterstained with Hoechst (blue). Scale bar = 10μm.</p

Kif4 protein expression increases with maternal ageing.

<p>Fluorescent immunolocalization of Kif4 (grey) in GV, MI and MII oocytes from young and maternally aged animals. Graphical representation of normalized Kif4 fluorescence at GV (p = 0.6891, Student’s <i>t</i>-test), MI (p = 0.0025, Student’s <i>t</i>-test) and MII (p<0.0001, Student’s <i>t</i>-test). Box plots show mean (centerline) with box outline 25-75^th percentiles and whiskers 10-90^th percentiles; n = number of oocytes examined from 4 animals. DNA is counterstained with Hoechst (blue). Scale bar = 10μm.</p

Kif4 is essential for correct spindle formation and cytokinesis.

<p>(A) Fluorescent immunolocalization of Kif4 (grey) in control and knock-down (KD) oocytes. Graphical representation of normalized Kif4 fluorescence; p<0.0001, Mann-Whitney test. (B) Fluorescent immunolocalization of α-tubulin at MII in control and Kif4 KD. Kif4 KD oocytes were found to have a higher percentage of abnormal spindles (orange arrow) and chromosome misalignment (red arrow). Graphical representation of abnormal spindle percentages per group; p = 0.0017, Fishers Exact test. (C) Phase contrast images of control and Kif4 KD MII oocytes. Graphical representation of PB1 size between control and Kif4 KD oocytes; p<0.0001, Mann-Whitney test. Box plots show mean (centerline) with box outline 25-75^th percentiles and whiskers 10-90^th percentiles, bar graphs show mean. n = number of oocytes examined from 3 replicates. DNA is counterstained with Hoechst (blue), scale bar = 10μm.</p

Kif4 has dynamic localization throughout oocyte meiosis.

<p>(A) Fluorescent immunolocalization of Kif4 (grey) at GV, GVB, MI and MII. Inserts highlight the localization of Kif4 to the nuclear membrane (yellow arrows) and microtubules (green arrow). Secondary only controls reveal no nonspecific antibody binding. Scale bar = 10μm. (B) Fluorescent immunolocalization of Kif4 (grey/green) to the kinetochore at MI (red arrow). Kinetochores are counter labelled with the inner kinetochore marker ACA (red). Scale bar = 2μm. (C) Fluorescent immunolocalization of Kif4 throughout MI at 6hrs to 8.5hrs post milrinone wash-out. Scale bar = 10μm. DNA is counterstained with Hoechst (blue).</p