Abstract. Vitamin A is one of the micronutrients which have been implicated in cattle reproduction.In cattle, ingested vitamin A, mainly as β-carotene (BC) from forages and retinol ester from formula feed, is metabolized and transported to the oocytes and cumulus-granulosa cells in ovarian follicles through binding to various interacting molecules. The active form of vitamin A, retinoic acid (RA), functions as a regulator of gene expression in these targets. Early research showed the positive effects of vitamin A supplementation on bovine fertility in artificial insemination, and several studies on effects of vitamin A metabolites used in other artificial reproductive techniques (ART), including superovulation, ovum pick up, and in vitro maturation culture have provided evidence for the specific roles of vitamin A in oocyte cytoplasmic maturation (acquisition of developmental competence of oocytes during their meiotic maturation period for the embryonic development after fertilization). BC may enhance cytoplasmic maturation by its antioxidant properties which cannot be replaced by RA. Furthermore, RA may promote cytoplasmic maturation of bovine oocytes via its modulatory effects on the gene expression of gonadotrophin receptors, midkine, cyclooxygenase-2, and nitric oxide synthase in cumulus-granulosa cells. Key words: Vitamin A, Cattle, Oocyte, Cytoplasmic maturation (J. Reprod. Dev. 51: [23][24][25][26][27][28][29][30][31][32][33][34][35] 2005) itamin A is one of the fat-soluble vitamins and is well known to regulate development, cellular growth and differentiation, and tissue function [1,2]. Since the earliest research into vitamin A function in cattle reproduction [3,4], vitamin A and its metabolites (Fig. 1) have been tested in various artificial reproduction techniques (ART) in cattle. It has been found so far that vitamin A and its metabolites affect ovarian follicular growth [5] and steroidogenesis [6], oviductal and uterine environments [7,8], immune functions [9-11], oocyte maturation, and embryo and conceptus development [12].Oocytes are formed during fetal life and are arrested at the prophase stage of the first meiotic division. After puberty, the fully grown oocyte arrested at the first meiotic prophase, the germinal vesicle stage, in the last folliclular wave [13] within the estrous cycle resumes meiosis in response to a preovulatory luteinizing hormone (LH) surge and reaches the metaphase stage of the second meiotic division. This process is called oocyte maturation [14] and is considered to involve not only the resumption of meiosis (nuclear maturation) but also the acquisition of developmental competence ( c y t o p l a s m i c m a t u r a t i o n ) f o r e m b r y o n i c d e v e l o p m e n t a f t e r f e r t i l i z a t i o n [ 1 5 , 1 6 ] .

The methylation pathway, which consists of two metabolic cycles of nutrients, i.e., the methionine and folate cycles, generates S-adenosylmethionine, the methyl donor for the methylation of DNA and histones. Using reverse transcription-polymerase chain reaction, we examined the gene expression patterns of the methylation pathway enzymes during bovine oocyte maturation and preimplantation embryonic development up to the blastocyst stage. Bovine oocytes were demonstrated to have the mRNA of all methylation pathway enzymes examined, namely, methionine adenosyltransferase 1A (MAT1A), MAT2A, MAT2B, S-adenosylhomocysteine hydrolase (AHCY), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), betaine-homocysteine methyltransferase (BHMT), serine hydroxymethyltransferase 1 (SHMT1), SHMT2, and 5,10-methylenetetrahydrofolate reductase (MTHFR). All the transcripts were consistently expressed throughout all developmental stages, except for MAT1A, which was not detected from the 8-cell stage onward and BHMT, which was not detected in the 8-cell stage. Immunofluorescence analysis of MAT1A protein revealed the relatively higher expression in oocytes and early cleavage stage embryos up to the 8-cell stage compared with the morula and blastocyst stage. Further, to investigate the effects of methylation pathway disruption during the earliest stages of embryonic development, the effects of exogenous homocysteine on preimplantation development and DNA methylation of bovine embryos were investigated in vitro. As results, high concentrations of homocysteine induced hypermethylation of genomic DNA as well as developmental retardation in bovine embryos. These results provide a new insight into nutrient-sensitive epigenetic regulation and perturbation at the earliest stage of our life.

The aim of this study was to investigate whether apoptosis occurs in cumulus cells during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes (CEOs). The bovine CEOs obtained from ovaries from an abattoir were cultured for 24 h in IVM medium in the presence or absence of 10% (v/v) fetal bovine serum. The developmental competence of enclosed oocytes, as assessed by the development of the blastocyst after IVF, was significantly higher in the serum-treated group than in the control group. The morphological features of apoptosis that were analysed by orcein staining were hardly detectable in the cumulus cells at the start (0 h) of IVM, but were evident at the end (24 h) of IVM both in the control and serum-treated groups. Genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect apoptotic internucleosomal DNA fragmentation. DNA fragmentation was hardly detectable at the start of IVM, but increased in a time-dependent manner as the IVM culture proceeded. DNA fragmentation was not observed in the oocytes, indicating that fragmentation occurs in cumulus cells. The degree of fragmentation was lower in the serum-treated group compared with the control group. The LM-PCR analysis of DNA extracted from CEOs at 24 h of IVM, in which the DNA had been pretreated with Klenow enzyme or T4 DNA polymerase, revealed that the characteristic forms of the DNA ends generated during cumulus cell apoptosis were mainly 3'-overhangs and blunt ends. In conclusion, the results of the present study demonstrate that cumulus cells in bovine CEOs spontaneously undergo apoptosis during IVM. The degree of apoptosis may be correlated with the developmental competence of the enclosed oocytes.

Perivascular adipose tissue exhibits characteristics of active local inflammation, which contributes to the development of atherosclerotic disease as a complication of obesity/metabolic syndrome. However, the precise role of perivascular adipose tissue in the progression of abdominal aortic aneurysm remains unclear. To test the hypothesis that genetic deletion of angiotensin II type 1a (AT) receptor in perivascular visceral adipose tissue (VAT) can attenuate aortic aneurysm formation in apolipoprotein E-deficient (ApoE) mice, we performed adipose tissue transplantation experiments by using an angiotensin II-induced aneurysm murine model, in which we transplanted VAT from ApoE or ApoE AT donor mice onto the abdominal aorta of ApoE recipient mice. Compared with ApoE VAT transplantation, ApoE AT VAT transplantation markedly attenuated aortic aneurysm formation, macrophage infiltration, and gelatinolytic activity in the abdominal aorta. AT receptor activation led to the polarization of macrophages in perivascular VAT toward the proinflammatory phenotype. Moreover, osteopontin expression and gelatinolytic activity were considerably lower in ApoE AT perivascular VAT than in ApoE perivascular VAT, and angiotensin II-induced osteopontin secretion from adipocytes was eliminated after deletion of AT receptor in adipocytes. Notably, induction of macrophage migration by conditioned medium from angiotensin II-stimulated wild-type adipocytes was suppressed by treatment with an osteopontin-neutralizing antibody, and ApoE OPN VAT transplantation more potently attenuated aortic aneurysm formation than ApoE VAT transplantation. Our findings indicate a previously unrecognized effect of AT receptor in perivascular VAT on the pathogenesis of abdominal aortic aneurysm.

The Morris water maze test (MWM) is a useful tool to evaluate rodents’ spatial learning and memory, but the outcome is susceptible to various experimental conditions. Thigmotaxis is a commonly observed behavioral pattern which is thought to be related to anxiety or fear. This behavior is associated with prolonged escape latency, but the impact of its frequency in the early stage on the final outcome is not clearly understood. We analyzed swim path trajectories in male C57BL/6 mice with or without bilateral common carotid artery stenosis (BCAS) treatment. There was no significant difference in the frequencies of particular types of trajectories according to ischemic brain surgery. The mouse groups with thigmotaxis showed significantly prolonged escape latency and lower cognitive score on day 5 compared to those without thigmotaxis. As the next step, we made a convolutional neural network (CNN) model to recognize the swim path trajectories. Our model could distinguish thigmotaxis from other trajectories with 96% accuracy and specificity as high as 0.98. These results suggest that thigmotaxis in the early training stage is a predictive factor for impaired performance in MWM, and machine learning can detect such behavior easily and automatically.