Blastocysts derivation from somatic cell fusion with premature oocytes (prematuration somatic cell fusion)

This study was undertaken to investigate the development of immature oocytes after their fusion with male somatic cells expressing red fluorescence protein (RFP). RFP‐expressing cells were fused with immature oocytes, matured in vitro and then parthenogenetically activated. Somatic nuclei showed spindle formation, 1st polar body extrusion after in vitro maturation and protruded the 2nd polar body after parthenogenetic activation. RFP was expressed in the resultant embryos; two‐cell stage and blastocysts. Chromosomal analysis showed aneuploidy in 81.82% of the resulting blastocysts while 18.18% of the resulting blastocysts were diploid. Among eight RFP‐expressing blastocysts, Xist mRNAs was detected in six while Sry mRNA was detected in only one blastocyst. We propose “prematuration somatic cell fusion” as an approach to generate embryos using somatic cells instead of spermatozoa. The current approach, if improved, would assist production of embryos for couples where the male partner is sterile, however, genetic and chromosomal analysis of the resultant embryos are required before transfer to the mothers.     

丙泊酚复合地佐辛用于无痛取卵手术的临床观察

目的:探讨丙泊酚复合地佐辛在生殖科取卵手术中的安全性和有效性。方法:选取择期行取卵手术的的患者100例,随机分为丙泊酚复合芬太尼组(F组)和丙泊酚复合地佐辛组(D组),观察和比较两组患者麻醉前(T0)、手术开始时(T1)、10min(T2)和手术结束时刻(T3)的呼吸、血压、心率、SPO_2的变化情况,意识丧失和唤醒时间,丙泊酚用量和不良反应的发生情况。对记录的数据进行组间比较,计量资料采用x±s,采用t检验,计数资料采用x2检验。结果:两组患者给药前后不同时点的心率、血压变化和意识丧失时间比较均无统计学差异,但D组丙泊酚用量低于F组,唤醒时间短于F组,F组疼痛和恶心的发生率高于D组,差异具有统计学意义(P0.05)。结论:丙泊酚复合地佐辛在生殖科取卵手术中可以提供相对较好的麻醉效果。     

Roles of actin binding proteins in mammalian oocyte maturation and beyond

Actin nucleation factors, which promote the formation of new actin filaments, have emerged in the last decade as key regulatory factors controlling asymmetric division in mammalian oocytes. Actin nucleators such as formin-2, spire, and the ARP2/3 complex have been found to be important regulators of actin remodeling during oocyte maturation. Another class of actin-binding proteins including cofilin, tropomyosin, myosin motors, capping proteins, tropomodulin, and Ezrin-Radixin-Moesin proteins are thought to control actin cytoskeleton dynamics at various steps of oocyte maturation. In addition, actin dynamics controlling asymmetric-symmetric transitions after fertilization is a new area of investigation. Taken together, defining the mechanisms by which actin-binding proteins regulate actin cytoskeletons is crucial for understanding the basic biology of mammalian gamete formation and pre-implantation development.     

Unwanted Horse Population in Illinois: Perceptions of Horse Owners,Non-Horse Owners,and Equine Industry Stakeholders

This paper presents the results of an investigation to determine perceptions, awareness, and knowledge of the unwanted horse population in Illinois from the viewpoint of horse owners, non-horse owners, and equine industry stakeholders. A questionnaire included items that pertained to knowledge of current legislation, equine background, current methods of controlling the unwanted horse population, and methods that respondents believe would reduce the unwanted horse population in Illinois. Results indicated that 58% of horse owners viewed horses as companion animals. Respondents perceived financial hardship to be the major reason why horses become unwanted. Current methods of managing unwanted horse populations were found to be ineffective. Reducing the costs of euthanasia and carcass disposal, allowing processing facilities to reopen in Illinois, and increasing the availability of gelding programs emerged as the most effective ways to manage the unwanted horse population. Results of this survey may lead to greater awareness of the unwanted horse population in Illinois. Furthermore, these results may lead to discussions about future legislation in the State designed to support and manage unwanted horses.     

TP53BP1 regulates chromosome alignment and spindle bipolarity in mouse oocytes

Meiotic oocytes lack classic centrosomes; therefore, bipolar spindle assembly depends on the clustering of acentriolar microtubule‐organizing centers (MTOCs) into two poles. The bipolar spindle is an essential cellular component that ensures accurate chromosome segregation during anaphase. If the spindle does not form properly, it can result in aneuploidy or cell death. However, the molecular mechanism by which the bipolar spindle is established is not yet fully understood. Tumor suppressor p53‐binding protein 1 (TP53BP1) is known to mediate the DNA damage response. Several recent studies have indicated that TP53BP1 has noncanonical roles in processes, such as spindle formation; however, the role of TP53BP1 in oocyte meiosis is currently unclear. Our results show that TP53BP1 knockdown affects spindle bipolarity and chromatin alignment by altering MTOC stability during oocyte maturation. TP53BP1 was localized in the cytoplasm and displayed an irregular cloud pattern around the spindle/chromosome region. TP53BP1 was also required for the correct localization of MTOCs into the two spindle poles during pro‐meiosis I. TP53BP1 deletion altered the MTOC‐localized Aurora Kinase A. TP53BP1 knockdown caused the microtubules to detach from the kinetochores and increased the rate of aneuploidy. Taken together, our data show that TP53BP1 plays crucial roles in chromosome stability and spindle bipolarity during meiotic maturation.     

Acentriolar microtubule organization centers and Ran‐mediated microtubule formation pathways are both required in porcine oocytes

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran‐mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC‐ and Ran‐mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo‐like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the Ran^GTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC‐ and Ran‐mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.     

All‐trans retinoic acid exposure increases connexin 43 expression in cumulus cells and improves embryo development in bovine oocytes

In developing follicles, cellular coupling within cumulus–oocyte complexes (COCs) creates a functional syncytium allowing for the passage of small molecules. In many species, intercellular coupling between granulosa cells results from the expression of connexin 43 (CX43 or Gja1) and the formation of gap junctional plaques. Previously, our lab has shown that oocytes with a higher developmental potential had higher CX43 expression in their cumulus cells compared with developmentally incompetent oocytes. All‐trans retinoic acid (ATRA) has been shown to increase CX43 expression in several different cell types. In this study we investigated the effect of ATRA treatment, during maturation, on CX43 expression and localization in cumulus cells and the developmental competence of bovine oocytes. COCs and granulosa cells exposed to ATRA during maturation had significantly higher CX43 expression and increased gap junctional coupling, respectively. In addition, there was a significant increase in the maturation, cleavage, and blastocyst rates in ATRA treated COCs. Data from these studies suggest that not only can CX43 be used as a biomarker for oocyte health, it can also potentially be manipulated using ATRA to increase the number of oocytes achieving developmental competence.     

Anaerobic fungal communities differ along the horse digestive tract

Anaerobic fungi are potent fibre degrading microbes in the equine hindgut, yet our understanding of their diversity and community structure is limited to date. In this preliminary work, using a clone library approach we studied the diversity of anaerobic fungi along six segments of the horse hindgut: caecum, right ventral colon (RVC), left ventral colon (LVC), left dorsal colon (LDC), right dorsal colon (RDC) and rectum. Of the 647 ITS1 clones, 61.7 % were assigned to genus level groups that are so far without any cultured representatives, and 38.0 % were assigned to the cultivated genera Neocallimastix (35.1 %), Orpinomyces (2.3 %), and Anaeromyces (0.6 %). AL1 dominated the group of uncultured anaerobic fungi, particularly in the RVC (88 %) and LDC (97 %). Sequences from the LSU clone library analysis of the LDC, however, split into two distinct phylogenetic clusters with low sequence identity to Caecomyces sp. (94–96 %) and Liebetanzomyces sp. (92 %) respectively. Sequences belonging to cultured Neocallimastix spp. dominated in LVC (81 %) and rectum (75.5 %). Quantification of anaerobic fungi showed significantly higher concentrations in RVC and RDC compared to other segments, which influenced the interpretation of the changes in anaerobic fungal diversity along the horse hindgut. These preliminary findings require further investigation.     

Fertilization causes a single Ca2+ increase that fully depends on Ca2+ influx in oocytes of limpets (Phylum Mollusca, Class Gastropoda)

Mature limpet oocytes arrested at the first metaphase (MI) of meiosis are activated by the stimulation of fertilizing sperm. The aim of the present study was to clarify the spatiotemporal property and mechanism of intracellular Ca2+ increase in limpet oocytes, which is a prerequisite signal for initiation of development at fertilization. In all of the five limpet species tested, the initial Ca2+ rising phase just after fertilization took the form of a centripetal Ca2+ wave spreading from the whole cortex to the center (cortical flash), yielding a homogeneous Ca2+ elevation throughout the oocyte. The Ca2+ level remained high during the subsequent plateau phase lasting for several minutes and then returned nearly to the original value. No additional Ca2+ increase followed the plateau phase at least by the time of first cleavage. Both rising and plateau phases of Ca2+ increase at fertilization were inhibited by removal of external Ca2+, suggesting that continuous Ca2+ entry occurs throughout the Ca2+ increase. Injection of inositol 1,4,5-trisphosphate (IP3) was effective in generating a Ca2+ increase in mature limpet oocytes arrested at MI; however, their ability to show an IP3-induced Ca2+ increase was extremely low, as compared with other animals. Responsiveness to IP3 injection in immature oocytes arrested at the first prophase (PI) was similar to that in the mature oocytes, suggesting that the IP3-induced Ca2+ release system does not develop during the process of meiotic maturation in limpet oocytes. Caffeine, cyclic adenosine diphosphate ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), the agents known to stimulate internal Ca2+ release mechanisms distinct from an IP3-dependent pathway, had no effect on intracellular Ca2+ changes in mature limpet oocytes. Labeling of the endoplasmic reticulum (ER) with DiI revealed that cortical ER clusters are only present in the localized region around meiotic chromosomes in mature oocytes. These data strongly suggest that Ca2+ release and its propagating mechanisms are undeveloped in limpet oocytes and that Ca2+ influx is the only Ca2+-mobilizing system available and functioning at fertilization.     

Ovarian dynamics, egg size, and egg number in relation to temperature and mating status in a butterfly

Although the temperature‐size rule, that is, an increase in egg (and body) size at lower temperatures, applies almost universally to ectotherms, the developmental mechanisms underlying this consistent pattern of phenotypic plasticity are hitherto unknown. By investigating ovarian dynamics and reproductive output in the tropical butterfly Bicyclus anynana (Butler) (Lepidoptera: Nymphalidae: Satyrinae) in relation to oviposition temperature and mating status, we tested the relevance of several competing hypotheses for temperature‐mediated variation in egg size and number. As expected, females ovipositing at a lower temperature laid fewer but larger eggs than those ovipositing at a higher temperature. Despite pronounced differences in egg‐laying rates, oocyte numbers were equal across temperatures at any given time, while oocyte size increased at the lower temperature. In contrast, there were greatly reduced oocyte numbers in mated compared to virgin females. Our results indicated that temperature‐mediated plasticity in egg size cannot be explained by reduced costs of somatic maintenance at lower temperatures, enabling the allocation of more resources to reproduction (reproductive investment was higher at the higher temperature). Furthermore, there was no indication for delayed oviposition (no accumulation of oocytes at the lower temperature, in contrast to virgin females). Rather, low temperatures greatly reduced the oocyte production (i.e., differentiation) rate and prolonged egg‐maturation time, causing low egg‐laying rates. Our data thus suggested that oocyte growth is less sensitive to temperature than oocyte production, resulting in a lower number of larger eggs at lower temperatures.