The Lon protease temporally restricts polar cell differentiation events during the Caulobacter cell cycle.

The highly conserved protease Lon has important regulatory and protein quality control functions in cells from the three domains of life. Despite many years of research on Lon, only a few specific protein substrates are known in most organisms. Here, we used a quantitative proteomics approach to identify novel substrates of Lon in the dimorphic bacterium Caulobacter crescentus. We focused our study on proteins involved in polar cell differentiation and investigated the developmental regulator StaR and the flagella hook length regulator FliK as specific Lon substrates in detail. We show that Lon recognizes these proteins at their C-termini, and that Lon-dependent degradation ensures their temporally restricted accumulation in the cell cycle phase when their function is needed. Disruption of this precise temporal regulation of StaR and FliK levels in a Δlon mutant contributes to defects in stalk biogenesis and motility, respectively, revealing a critical role of Lon in coordinating developmental processes with cell cycle progression. Our work underscores the importance of Lon in the regulation of complex temporally controlled processes by adjusting the concentrations of critical regulatory proteins. Furthermore, this study includes the first characterization of FliK in C. crescentus and uncovers a dual role of the C-terminal amino acids of FliK in protein function and degradation.

Two mutations in TUBB8 cause developmental arrest in human oocytes and early embryos.

RESEARCH QUESTION: How can the effect of genetic mutations that may cause primary female infertility be evaluated? DESIGN: Patients and their family members underwent whole-exome sequencing and Sanger sequencing to detect the infertility-causing gene and inheritance pattern. To study the function of mutant proteins in vitro, vectors containing wild-type or mutant TUBB8 cDNA were constructed for transient expression in HeLa cells, and in-vitro transcribed mRNA were used for microinjection in germinal vesicle-stage mouse oocytes. Immunofluorescence staining was used to observe the microtubule structure in HeLa cells or meiotic spindle in mouse oocytes. RESULTS: A maternally inherited TUBB8 (Tubulin beta 8 class VIII) mutation (NM_177987.2: c. 959G>A: p. R320H) and a previously reported (NM_177987.2: c. 161C>T: p. A54V) recessive mutation from two infertile female patients were identified. The oocytes from the patient carrying p.A54V mutation failed fertilization, whereas oocytes with p.R320H mutation could be fertilized but showed heavy fragmentation during early development. In vitro, functional assays showed that p. A54V mutant disrupted the microtubule structure in HeLa cells (49.3% of transfected cells) and caused large polar body extrusion in mouse oocytes (27.5%), whereas the p.R320H mutant caused a higher abnormal rate (69.7%) in cultured cells and arrested mouse oocytes at meiosis I (38.7%). CONCLUSION: Two TUBB8 mutations (p.A54V and p.R320H) were identified and their pathogeny was confirmed by in-vitro functional assays.

TUBB8 Mutations Cause Female Infertility with Large Polar Body Oocyte and Fertilization Failure.

Tubulin beta 8 class VIII (TUBB8) is a special ß-tubulin isotype that mainly expressed in primate oocytes and early embryos and identified as the disease-causing gene of human oocyte maturation arrest. To identify the disease-causing genes in 2 patients with female infertility due to large polar body oocyte or fertilization failure, whole-exome sequencing was performed on the patients and available family members. We identified a novel heterozygous missense mutation c.817C>G (p.L273V) and a recently reported heterozygous missense mutation c.608A>G (p.D203G) in TUBB8 from two patients, respectively. We found oocyte with a large polar body in the patient who carried the p.D203G mutation in TUBB8. Bioinformatics analysis showed that these two mutations are harmful. The results of western blot and RT-PCR experiments showed that the D203G mutation caused a significant decrease in the expression of TUBB8, and immunostaining showed that the TUBB8 mutation caused abnormal microtubule morphology. These findings suggest that TUBB8 mutations resulted in oocyte with a large polar body and fertilization failure in patients.

Detection of the Polar Body After Fertilization.

The polar body, with haploid DNA, is a small cell produced during the meiosis of an oocyte. Here, we describe the detailed procedures for the detection of the second polar body in zebrafish (Danio rerio) embryos after 10 min post fertilization. A polar body can be easily distinguished as a small dot with a DAPI-stained nucleus surrounded by Phalloidin-labeled F-actin in each fertilized zebrafish embryo.

Role of PB1 Midbody Remnant Creating Tethered Polar Bodies during Meiosis II.

Polar body (PB) formation is an extreme form of unequal cell division that occurs in oocytes due to the eccentric position of the small meiotic spindle near the oocyte cortex. Prior to PB formation, a chromatin-centered process causes the cortex overlying the meiotic chromosomes to become polarized. This polarized cortical subdomain marks the site where a cortical protrusion or outpocket forms at the oocyte surface creating the future PBs. Using ascidians, we observed that PB1 becomes tethered to the fertilized egg via PB2, indicating that the site of PB1 cytokinesis directed the precise site for PB2 emission. We therefore studied whether the midbody remnant left behind following PB1 emission was involved, together with the egg chromatin, in defining the precise cortical site for PB2 emission. During outpocketing of PB2 in ascidians, we discovered that a small structure around 1 µm in diameter protruded from the cortical outpocket that will form the future PB2, which we define as the "polar corps". As emission of PB2 progressed, this small polar corps became localized between PB2 and PB1 and appeared to link PB2 to PB1. We tested the hypothesis that this small polar corps on the surface of the forming PB2 outpocket was the midbody remnant from the previous round of PB1 cytokinesis. We had previously discovered that Plk1::Ven labeled midbody remnants in ascidian embryos. We therefore used Plk1::Ven to follow the dynamics of the PB1 midbody remnant during meiosis II. Plk1::Ven strongly labeled the small polar corps that formed on the surface of the cortical outpocket that created PB2. Following emission of PB2, this polar corps was rich in Plk1::Ven and linked PB2 to PB1. By labelling actin (with TRITC-Phalloidin) we also demonstrated that actin accumulates at the midbody remnant and also forms a cortical cap around the midbody remnant in meiosis II that prefigured the precise site of cortical outpocketing during PB2 emission. Phalloidin staining of actin and immunolabelling of anti-phospho aPKC during meiosis II in fertilized eggs that had PB1 removed suggested that the midbody remnant remained within the fertilized egg following emission of PB1. Dynamic imaging of microtubules labelled with Ens::3GFP, MAP7::GFP or EB3::3GFP showed that one pole of the second meiotic spindle was located near the midbody remnant while the other pole rotated away from the cortex during outpocketing. Finally, we report that failure of the second meiotic spindle to rotate can lead to the formation of two cortical outpockets at anaphase II, one above each set of chromatids. It is not known whether the midbody remnant of PB1 is involved in directing the precise location of PB2 since our data are correlative in ascidians. However, a review of the literature indicates that PB1 is tethered to the egg surface via PB2 in several species including members of the cnidarians, lophotrochozoa and echinoids, suggesting that the midbody remnant formed during PB1 emission may be involved in directing the precise site of PB2 emission throughout the invertebrates.

Evaluating the value of day 0 of an ICSI cycle on indicating laboratory outcome.

A number of oocyte characteristics have been associated with fertilization, implantation and live-birth rates, albeit without reaching a consensus. This study aims to delineate possible associations between oocyte characteristics, oocyte behavior during intracytoplasmic sperm injection (ICSI), fertilization potential, and laboratory outcomes. Four-hundred and seventy-seven patients, yielding 3452 oocytes, were enrolled in this prospective observational study from 2015 to 2018. Οoplasm granularity was associated with poor embryo quality and higher probabilities of post-ICSI oocytes and embryos discarded in any developmental stage and never selected for embryo transfer or cryopreservation (p < 0.001). Both sudden or difficult ooplasm aspiration, and high or lack of resistance during ICSI were associated with either a poor Zygote-Score or fertilization failure (p < 0.001). Sudden or difficult ooplasm aspiration and high resistance during ICSI penetration were positively associated with resulting to a post-ICSI oocyte or embryo that would be selected for discard. Evaluation of oocyte characteristics and oocyte behavior during ICSI may provide early information regarding laboratory and cycle outcomes. Particularly, ooplasm granularity, and fragmentation of polar body, along with sudden or difficult ooplasm aspiration and high or lack of resistance during ICSI penetration may hinder the outcome of an ICSI cycle. The associations presented herein may contribute towards development of a grading system or a prediction model. Taking into account information on oocytes and ICSI behavior may effectively assist in enhancing IVF outcome rates.

Time of first polar body extrusion affects the developmental competence of equine oocytes after intracytoplasmic sperm injection.

Assisted reproduction techniques (ARTs) have become widespread in the equine breeding industry. In particular, the combination of oocyte recovery from live mares followed by IVM and intracytoplasmic sperm injection (ICSI) has increased markedly among the ARTs used with valuable or low-fertility animals. There is currently no consensus among research groups regarding the optimal oocyte maturation period to produce high-quality embryos. In this study, we report the maturation dynamics of equine oocytes at different time points, from 20 to 40h (Experiment 1). In addition, in Experiment 2, equine ICSI blastocysts were produced from oocytes that exhibited early (up to 24h) or late (28-30h) extrusion of the first polar body (PB). Blastocyst rates and diameter were recorded and embryo quality was assessed by analysing the number of apoptotic cells and Yes-associated protein 1 (YAP1) expression. By 20h of IVM, 42% of oocytes were mature, and the remaining oocytes matured within the next 17h of IVM. Although no differences were found in cell apoptosis or the number of YAP1-positive cells between groups exhibiting early and late PB extrusion, embryos from the early group (Group I) exhibited an improved total cell number and blastocyst rate compared to embryos from the late group (Group II) (18.60% vs 10.17% respectively).

Fertilization and Cleavage Axes Differ In Primates Conceived By Conventional (IVF) Versus Intracytoplasmic Sperm Injection (ICSI).

With nearly ten million babies conceived globally, using assisted reproductive technologies, fundamental questions remain; e.g., How do the sperm and egg DNA unite? Does ICSI have consequences that IVF does not? Here, pronuclear and mitotic events in nonhuman primate zygotes leading to the establishment of polarity are investigated by multidimensional time-lapse video microscopy and immunocytochemistry. Multiplane videos after ICSI show atypical sperm head displacement beneath the oocyte cortex and eccentric para-tangential pronuclear alignment compared to IVF zygotes. Neither fertilization procedure generates incorporation cones. At first interphase, apposed pronuclei align obliquely to the animal-vegetal axis after ICSI, with asymmetric furrows assembling from the male pronucleus. Furrows form within 30° of the animal pole, but typically, not through the ICSI injection site. Membrane flow drives polar bodies and the ICSI site into the furrow. Mitotic spindle imaging suggests para-tangential pronuclear orientation, which initiates random spindle axes and minimal spindle:cortex interactions. Parthenogenetic pronuclei drift centripetally and assemble astral spindles lacking cortical interactions, leading to random furrows through the animal pole. Conversely, androgenotes display cortex-only pronuclear interactions mimicking ICSI. First cleavage axis determination in primates involves dynamic cortex-microtubule interactions among male pronuclei, centrosomal microtubules, and the animal pole, but not the ICSI site.

Chromatin organization and timing of polar body I extrusion identify developmentally competent mouse oocytes.

In the mouse, the use of the DNA-binding fluorochrome Hoechst 33342 allows the classification of fully-grown antral oocytes into two categories distinguished by their chromatin conformation: surrounding nucleolus (SN) and not-surrounding nucleolus (NSN) oocytes, the former capable of completing development, the latter unable to proceed beyond the 2-cell stage. In the present study, time-lapse observation of SN and NSN oocyte GV-to-MII transition highlighted differences in the timing of germinal vesicle breakdown (GVBD) and polar body I (PB-I) extrusion. PB-I extrusion, but not GVBD, revealed the presence of three main groups of significantly different oocytes: Group A (456-576 min) comprising mainly SN oocytes (91.4%), group B (584-728 min) entailing an almost equivalent percentage of SN (52.7%) and NSN (47.3%) oocytes, whereas group C (736-896 min) consisting of almost all NSN (94.4%) oocytes. In a further set of time-lapse experiments, GV oocytes were in vitro matured without Hoechst staining and, depending on the timing of PB-I extrusion, sorted into group A, B or C, inseminated with sperm and observed throughout preimplantation. The results show that 26.2 ± 12.3% of group A, 2.4 ± 5.0% of group B and none of group C MII oocytes developed to blastocyst. Overall, this study shows that SN oocytes that complete MI earlier are those with a better developmental competence. The possibility to avoid the use of the invasive DNA-binding fluorochrome Hoechst is relevant for future applications in human and domestic animal reproductive technologies.

RAB8A GTPase regulates spindle migration and Golgi apparatus distribution via ROCK-mediated actin assembly in mouse oocyte meiosis†.

Actin filaments are widely involved in multiple cellular processes in oocyte meiosis, such as spindle migration and polar body extrusion. The actin nucleators like Arp2/3 complex and formins are the most recognized molecules for actin assembly in oocytes. In the present study, we report that the vesicle trafficking factor, RAB8A GTPase, is a new regulator critical for actin assembly in meiosis. Our results showed that RAB8A was localized at both the spindle periphery and cortex in mouse oocytes, which was similar to the localization patterns of actin filaments. RAB8A depletion caused spindle migration defects and the failure of polar body extrusion, which could have been due to decreases in both cytoplasmic and cortical actin filaments in oocytes. Based on mass spectrometry analysis, we showed that RAB8A promoted actin assembly through its modulation on the ROCK-LIMK signaling pathway. Moreover, we demonstrated that RAB8A colocalized and interacted with GM130 at the spindle periphery and that RAB8A depletion caused the disruption of GM130-docked Golgi distribution. Taken together, our data indicated that RAB8A was required for Golgi distribution, spindle migration, and polar body extrusion via ROCK-mediated actin assembly in mouse oocyte meiosis.