Extracellular Vesicles Contribute to the Difference in Lipid Composition between Ovarian Follicles of Different Size Revealed by Mass Spectrometry Imaging.

Follicular fluid (FF) ensures a safe environment for oocyte growth and maturation inside the ovarian follicle in mammals. In each cycle, the large dominant follicle (LF) contains the oocyte designated to be ovulated, whereas the small subordinate follicles (SFs) of the same wave will die through atresia. In cows, the oocytes from the SF, being 2 mm in size, are suitable for in vitro reproduction biotechnologies, and their competence in developing an embryo depends on the size of the follicles. FF contains proteins, metabolites, fatty acids, and a multitude of extracellular vesicles (ffEVs) of different origins, which may influence oocyte competence through bidirectional exchanges of specific molecular cargo between follicular cells and enclosed oocytes. FF composition evolves along with follicle growth, and the abundance of different lipids varies between the LF and SF. Here, significant differences in FF lipid content between the LFs and SFs within the same ovary were demonstrated by MALD-TOF mass spectrometry imaging on bovine ovarian sections. We then aimed to enlighten the lipid composition of FF, and MALDI-TOF lipid profiling was performed on cellular, vesicular, and liquid fractions of FF. Differential analyses on the abundance of detected lipid features revealed specific enrichment of phospholipids in different ffEV types, such as microvesicles (MVs) and exosomes (Exo), compared to depleted FF. MALDI-TOF lipid profiling on MVs and Exo from the LF and SF samples (n = 24) revealed that more than 40% of detected features were differentially abundant between the groups of MVs and Exo from the different follicles (p < 0.01, fold change > 2). Glycerophospholipid and sphingolipid features were more abundant in ffEVs from the SFs, whereas different lysophospholipids, including phosphatidylinositols, were more abundant in the LFs. As determined by functional analysis, the specific lipid composition of ffEVs suggested the involvement of vesicular lipids in cell signaling pathways and largely contributed to the differentiation of the dominant and subordinate follicles.

Centrosome Formation in the Bovine Early Embryo.

Centrosome formation during early development in mice and rats occurs due to the appearance of centrioles de novo. In contrast, in humans and other non-rodent mammals, centrioles are thought to be derived from spermatozoa. Ultrastructural study of zygotes and early embryos of cattle at full series of ultrathin sections show that the proximal centriole of the spermatozoon disappears by the end of the first cleavage division. Centrioles appear in two to four cell embryos in fertilized oocytes and in parthenogenetic embryos. Centriole formation includes the appearance of atypical centrioles with randomly arranged triplets and centrioles with microtubule triplets of various lengths. After the third cleavage, four centriolar cylinders appear for the first time in the blastomeres while each embryo still has two atypical centrioles. Our results showed that the mechanisms of centriole formation in different groups of mammals are universal, differing only in the stage of development in which they occur.

Protein Palmitoylation in Bovine Ovarian Follicle.

Protein palmitoylation is a reversible post-translational modification by fatty acids (FA), mainly a palmitate (C16:0). Palmitoylation allows protein shuttling between the plasma membrane and cytosol to regulate protein stability, sorting and signaling activity and its deficiency leads to diseases. We aimed to characterize the palmitoyl-proteome of ovarian follicular cells and molecular machinery regulating protein palmitoylation within the follicle. For the first time, 84 palmitoylated proteins were identified from bovine granulosa cells (GC), cumulus cells (CC) and oocytes by acyl-biotin exchange proteomics. Of these, 32 were transmembrane proteins and 27 proteins were detected in bovine follicular fluid extracellular vesicles (ffEVs). Expression of palmitoylation and depalmitoylation enzymes as palmitoyltransferases (ZDHHCs), acylthioesterases (LYPLA1 and LYPLA2) and palmitoylthioesterases (PPT1 and PPT2) were analysed using transcriptome and proteome data in oocytes, CC and GC. By immunofluorescence, ZDHHC16, PPT1, PPT2 and LYPLA2 proteins were localized in GC, CC and oocyte. In oocyte and CC, abundance of palmitoylation-related enzymes significantly varied during oocyte maturation. These variations and the involvement of identified palmitoyl-proteins in oxidation-reduction processes, energy metabolism, protein localization, vesicle-mediated transport, response to stress, G-protein mediated and other signaling pathways suggests that protein palmitoylation may play important roles in oocyte maturation and ffEV-mediated communications within the follicle.

Delaying Effects of Prolactin and Growth Hormone on Aging Processes in Bovine Oocytes Matured In Vitro.

Aging processes accelerate dramatically in oocytes that have reached the metaphase-II (M-II) stage. The present work aimed to study the patterns and intracellular pathways of actions of prolactin (PRL) and growth hormone (GH) on age-associated changes in bovine M-II oocytes aging in vitro. To this end, we analyzed spontaneous parthenogenetic activation (cytogenetic assay), apoptosis (TUNEL assay), and the developmental capacity (IVF/IVC) of in vitro-matured oocytes after prolonged culturing. Both PRL and GH reduced the activation rate of aging cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs), and their respective hormone receptors were revealed in the ova. The inhibitor of Src-family tyrosine kinases PP2 eliminated the effects of PRL and GH on meiotic arrest in DOs, whereas the MEK inhibitor U0126 only abolished the PRL effect. Furthermore, PRL was able to maintain the apoptosis resistance and developmental competence of aging CEOs. The protein kinase C inhibitor calphostin C suppressed both the actions of PRL. Thus, PRL and GH can directly support meiotic arrest in aging M-II oocytes by activating MAP kinases and/or Src-family kinases. The effect of PRL in maintaining the developmental capacity of aging oocytes is cumulus-dependent and related to the pro-survival action of the protein kinase C-mediated signal pathway.

Prolactin and growth hormone affect metaphase-II chromosomes in aging oocytes via cumulus cells using similar signaling pathways.

General senescence of the adult organism is closely connected with reproductive one. Meanwhile, the age-related reduction in the female fertility is primarily associated with a decline in the gamete quality. Molecular and cellular changes in oocytes of old mammalian females are very similar to those occurring during aging of matured ova of their young counterparts, suggesting similarities in underlying mechanisms. The aim of the present work was to study actions of two related pituitary hormones, prolactin (PRL) and growth hormone (GH), on age-associated modifications of metaphase-II (M-II) chromosomes in bovine oocytes using a model of the prolonged culture. We analyzed: (1) effects of PRL and GH on abnormal changes in the chromosome morphology in aging matured oocytes and the role of cumulus cells in these effects and (2) signaling pathways involved in the hormone actions. During the prolonged culture of oocytes, a gradual rise in the frequency of destructive modifications of M-II chromosomes was revealed. In the case of cumulus-enclosed oocytes (CEOs), PRL and GH exerted dose-dependent biphasic effects on the frequency of these modifications. Both PRL (50 ng/ml) and GH (10 ng/ml) decelerated the abnormal chromosome changes in CEOs, but did not affect the chromosome configuration in denuded oocytes. Concurrently, the presence of PRL and GH receptors in cumulus cells surrounding matured oocytes was demonstrated. Attenuating effects of both hormones on the chromosome modifications in aging CEOs were abolished by PP2 (an inhibitor of Src-family tyrosine kinases), triciribine (an inhibitor of Akt kinase), and calphostin C (a protein kinase C inhibitor). Our findings indicate that PRL and GH can exert the similar decelerating action on age-associated alterations in the M-II chromosome morphology in bovine ova, which is mediated by cumulus cells and may be related to activation of Src-family tyrosine kinases as well as Akt- and protein kinase C-dependent signal pathways.

Co-expression of the Thermotoga neapolitana aglB gene with an upstream 3'-coding fragment of the malG gene improves enzymatic characteristics of recombinant AglB cyclomaltodextrinase.

A cluster of Thermotoga neapolitana genes participating in starch degradation includes the malG gene of sugar transport protein and the aglB gene of cyclomaltodextrinase. The start and stop codons of these genes share a common overlapping sequence, aTGAtg. Here, we compared properties of expression products of three different constructs with aglB from T. neapolitana. The first expression vector contained the aglB gene linked to an upstream 90-bp 3'-terminal region of the malG gene with the stop codon overlapping with the start codon of aglB. The second construct included the isolated coding sequence of aglB with two tandem potential start codons. The expression product of this construct in Escherichia coli had two tandem Met residues at its N terminus and was characterized by low thermostability and high tendency to aggregate. In contrast, co-expression of aglB and the 3'-terminal region of malG (the first construct) resulted in AglB with only one N-terminal Met residue and a much higher specific activity of cyclomaltodextrinase. Moreover, the enzyme expressed by such a construct was more thermostable and less prone to aggregation. The third construct was the same as the second one except that it contained only one ATG start codon. The product of its expression had kinetic and other properties similar to those of the enzyme with only one N-terminal Met residue.