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The Journal of Reproduction and... Dec 2017In embryo transfer experiments in mice, pseudopregnant females as recipients are prepared by sterile mating with vasectomized males. Because only females at the...
In embryo transfer experiments in mice, pseudopregnant females as recipients are prepared by sterile mating with vasectomized males. Because only females at the proestrus stage accept males, such females are selected from a stock of animals based on the appearance of their external genital tract. Therefore, the efficiency of preparing pseudopregnant females largely depends on the size of female colonies and the skill of the operators who select females for sterile mating. In this study, we examined whether the efficiency of preparing pseudopregnant females could be improved by applying an estrous cycle synchronization method by progesterone (P4) pretreatment, which significantly enhances the superovulation outcome in mice. We confirmed that after two daily injections of P4 (designated Days 1 and 2) in randomly selected females, the estrous cycles of most females (about 85%) were synchronized at metestrus on Day 3. When P4-treated females were paired with vasectomized males for 4 days (Days 4-8), a vaginal plug was found in 63% (20/32) of the females on Day 7. After the transfer of vitrified-warmed embryos into their oviducts, 52% (73/140) of the embryos successfully developed into offspring, the rate being comparable to that of the conventional embryo transfer procedure. Similarly, 77% (24/31) of females became pregnant by fertile mating with intact males for 3 days, which allowed the scheduled preparation of foster mothers. Thus, our estrous cycle synchronization method may omit the conventional experience-based process of visually observing the vagina to choose females for embryo transfer. Furthermore, it is expected that the size of female stocks for recipients can be reduced to less than 20%, which could be a great advantage for facilities/laboratories undertaking mouse-assisted reproductive technology.
Topics: Animals; Embryo Transfer; Estrus Synchronization; Female; Male; Pregnancy; Progesterone; Pseudopregnancy
PubMed: 28824024
DOI: 10.1262/jrd.2017-068 -
Frontiers in Cellular Neuroscience 2017The antero-ventral periventricular zone (AVPV) and medial preoptic area (MPOA) have been recognized as gonadal hormone receptive regions of the rodent brain that-via...
The antero-ventral periventricular zone (AVPV) and medial preoptic area (MPOA) have been recognized as gonadal hormone receptive regions of the rodent brain that-via wiring to gonadotropin-releasing hormone (GnRH) neurons-contribute to orchestration of the preovulatory GnRH surge. We hypothesized that neural genes regulating the induction of GnRH surge show altered expression in proestrus. Therefore, we compared the expression of 48 genes obtained from intact proestrous and metestrous mice, respectively, by quantitative real-time PCR (qPCR) method. Differential expression of 24 genes reached significance ( < 0.05). Genes upregulated in proestrus encoded neuropeptides (kisspeptin (KP), galanin (GAL), neurotensin (NT), cholecystokinin (CCK)), hormone receptors (growth hormone secretagogue receptor, μ-opioid receptor), gonadal steroid receptors (estrogen receptor alpha (ERα), progesterone receptor (PR), androgen receptor (AR)), solute carrier family proteins (vesicular glutamate transporter 2, vesicular monoamine transporter 2), proteins of transmitter synthesis (tyrosine hydroxylase (TH)) and transmitter receptor subunit (AMPA4), and other proteins (uncoupling protein 2, nuclear receptor related 1 protein). Proestrus evoked a marked downregulation of genes coding for adenosine A2a receptor, vesicular gamma-aminobutyric acid (GABA) transporter, 4-aminobutyrate aminotransferase, tachykinin precursor 1, NT receptor 3, arginine vasopressin receptor 1A, cannabinoid receptor 1, ephrin receptor A3 and aldehyde dehydrogenase 1 family, member L1. Immunocytochemistry was used to visualize the proteins encoded by and genes in neuronal subsets of the AVPV/MPOA of the proestrous mice. The results indicate that gene expression of the AVPV/MPOA is significantly modified at late proestrus including genes that code for neuropeptides, gonadal steroid hormone receptors and synaptic vesicle transporters. These events support cellular and neuronal network requirements of the positive estradiol feedback action and contribute to preparation of the GnRH neuron system for the pre-ovulatory surge release.
PubMed: 28725181
DOI: 10.3389/fncel.2017.00183 -
Journal of Toxicologic Pathology Apr 2017When conducting vaginal irritation studies, ovariectomized rats or rabbits are typically used according to practical reports. In the present study, we evaluated the...
When conducting vaginal irritation studies, ovariectomized rats or rabbits are typically used according to practical reports. In the present study, we evaluated the influence of the estrus cycle in a vaginal irritation study using intact rats and ovariectomized rats, which exhibit a late diestrus-like condition, to determine whether intact rats can be useful for evaluating vaginal irritancy. Rats were divided into 4 groups: proestrus, estrus, and metestrus or diestrus in intact rats and ovariectomized rats. All the rats in each group were treated with a vehicle or sodium dodecyl sulfate, as the irritant, in single-dose and 4-day repeat-dose vaginal irritation studies. Each rat's vagina was examined histopathologically, and the irritation score was calculated using a semiquantitative scoring system. In the single-dose study, the irritation scores for the proestrus or ovariectomized groups treated with sodium dodecyl sulfate were higher than those of the estrus group or metestrus or diestrus group. In the 4-day repeat-dose study, a significant histopathological difference was not found among the intact rats (proestrus, estrus, and metestrus or diestrus groups), and the irritation score range of the intact rats was similar to that of the ovariectomized rats, though the mean score of the intact rats was slightly lower than that of the ovariectomized rats. These results suggest that intact rats might be well suited for 4-day vaginal irritation studies and useful for evaluating vaginal irritancy using not only the mean score, but also individual irritation score ranges, whereas the estrus cycle would need to be identified in single-dose vaginal irritation studies.
PubMed: 28458454
DOI: 10.1293/tox.2016-0059 -
Reproductive Biology and Endocrinology... Apr 2017The synthesis of sex steroids is controlled by several enzymes such as17α-hydroxylase cytochrome P450 (P450c17) catalyzing androgen synthesis and aromatase cytochrome...
BACKGROUND
The synthesis of sex steroids is controlled by several enzymes such as17α-hydroxylase cytochrome P450 (P450c17) catalyzing androgen synthesis and aromatase cytochrome P450 (P450arom) catalyzing estrogen synthesis, both of which must complex with the redox partner NADPH-cytochrome P450 oxidoreductase (CPR) for activity. Previous studies have identified expression of steroidogenic enzymes in vaginal tissue, suggesting local sex steroid synthesis. The current studies investigate P450c17, P450aromatase and CPR expression in vaginal mucosa of Galea spixii (Spix cavy) by immuno-histochemical and western immunoblot analyses.
METHODS
Stages of estrous cyclicity were monitored by vaginal exfoliative cytology. After euthanasia, vaginal tissues were retrieved, fixed and frozen at diestrus, proestrus, estrus and metestrus. The ovaries and testis were used as positive control tissues for immunohistochemistry.
RESULTS
Data from cytological study allowed identification of different estrous cycle phases. Immunohistochemical analysis showed different sites of expression of steroidogenic enzymes along with tissue response throughout different phases of the estrous cycle. However, further studies are needed to characterize the derived hormones synthesized by, and the enzymes activities associated with, vaginal tissues.
CONCLUSION
Current results not only support the expression of enzymes involved in sex steroid synthesis in the wall of the vagina, they also indicate that expression changes with the stage of the cycle, both the levels and types of cells exhibiting expression. Thus, changes in proliferation of vaginal epithelial cells and the differentiation of the mucosa may be influenced by local steroid synthesis as well as circulating androgens and estrogens.
Topics: Animals; Cell Proliferation; Epithelium; Estrous Cycle; Female; Gonadal Steroid Hormones; Male; Rodentia; Vagina
PubMed: 28438170
DOI: 10.1186/s12958-017-0248-3 -
Veterinary Research Forum : An... 2016It was assumed that uterine stem cells are responsible for the unique regenerative capacity of uterine. Therefore, the aim of the present study was to investigate the...
It was assumed that uterine stem cells are responsible for the unique regenerative capacity of uterine. Therefore, the aim of the present study was to investigate the expression of the pluripotent stem cell markers in the mice uterine tissue during different stages of estrous cycles. Twelve virgin female NMRI mice (6 to 8 weeks old) were considered at proestrus, estrus, metestrus and diestrus according to the cell types observed in the vaginal smear and underwent hysterectomy operation. Quantitative real-time polymerase chain reaction (PCR) and immunohistochemical staining for pluripotent stem cell markers (SOX2, OCT4, KLF4, and NANOG) were performed. Immunofluorescence staining revealed that expression and localization of the pluripotency markers SOX2, OCT4, KLF4, and NANOG at the protein level were not different throughout estrous cycle. Also, mRNA of pluripotency markers was detected in all tested samples. However, there were no significant differences in their genes expression at each stage and during the estrous cycle. Different hormonal profile during the estrous cycle could not affect expression of pluripotent stem cell markers in uterine tissue.
PubMed: 27872713
DOI: No ID Found -
Neurologia (Barcelona, Spain) 2017Stereotactic surgery is used to place electrodes or cannulas in the brain in order to study the function of several brain structures in preclinical research. The...
INTRODUCTION
Stereotactic surgery is used to place electrodes or cannulas in the brain in order to study the function of several brain structures in preclinical research. The hippocampus has been extensively studied with this methodology due to its involvement in a wide range of neurological, cognitive, emotional, and affective disorders. However, the effects of stereotactic surgery on coordination and motor activity should be evaluated in order to determine whether this surgical procedure causes any neurological alterations that may bias the results of studies incorporating this technique.
METHODS
We evaluated the effects of stereotactic surgery and implantation of a cannula into the hippocampus of female Wistar rats on the motor activity, forced swim, and rotarod tests. The stage of the oestrous cycle was included in the statistical analysis.
RESULTS
Stereotactic surgery had no impact on any of the motor activity variables assessed in the open field (squares crossed, time spent in grooming, and rearing), forced swim (turning behaviour, lateral swimming, latency to first immobility, and time spent immobile), and rotarod (latency to fall) tests, compared with intact rats. Regardless of surgical manipulation, rats in the metestrus and diestrus stages crossed a greater number of squares and displayed longer immobility times than those in the proestrus and estrus stages.
CONCLUSION
Stereotactic surgery for cannula placement in the dorsal hippocampus does not affect coordination and motor activity in rats. We can therefore conclude that this procedure has no neurological complications that may interfere in the interpretation of results of studies applying this technique.
Topics: Animals; Cannula; Female; Hippocampus; Motor Activity; Postural Balance; Rats; Rats, Wistar; Stereotaxic Techniques; Swimming
PubMed: 27157521
DOI: 10.1016/j.nrl.2016.03.004 -
The Journal of Endocrinology Jul 2016Chemerin, a chemokine, plays important roles in immune responses, inflammation, adipogenesis, and carbohydrate metabolism. Our recent research has shown that chemerin...
Chemerin, a chemokine, plays important roles in immune responses, inflammation, adipogenesis, and carbohydrate metabolism. Our recent research has shown that chemerin has an inhibitory effect on hormone secretion from the testis and ovary. However, whether G protein-coupled receptor 1 (GPR1), the active receptor for chemerin, regulates steroidogenesis and luteolysis in the corpus luteum is still unknown. In this study, we established a pregnant mare serum gonadotropin-human chorionic gonadotropin (PMSG-hCG) superovulation model, a prostaglandin F2α (PGF2α) luteolysis model, and follicle and corpus luteum culture models to analyze the role of chemerin signaling through GPR1 in the synthesis and secretion of gonadal hormones during follicular/luteal development and luteolysis. Our results, for the first time, show that chemerin and GPR1 are both differentially expressed in the ovary over the course of the estrous cycle, with highest levels in estrus and metestrus. GPR1 has been localized to granulosa cells, cumulus cells, and the corpus luteum by immunohistochemistry (IHC). In vitro, we found that chemerin suppresses hCG-induced progesterone production in cultured follicle and corpus luteum and that this effect is attenuated significantly by anti-GPR1 MAB treatment. Furthermore, when the phosphoinositide 3-kinase (PI3K) pathway was blocked, the attenuating effect of GPR1 MAB was abrogated. Interestingly, PGF2α induces luteolysis through activation of caspase-3, leading to a reduction in progesterone secretion. Treatment with GPR1 MAB blocked the PGF2α effect on caspase-3 expression and progesterone secretion. This study indicates that chemerin/GPR1 signaling directly or indirectly regulates progesterone synthesis and secretion during the processes of follicular development, corpus luteum formation, and PGF2α-induced luteolysis.
Topics: Animals; Caspase 3; Chemokines; Chorionic Gonadotropin; Corpus Luteum; Dinoprost; Estradiol; Female; Intercellular Signaling Peptides and Proteins; Luteolysis; Mice; Phosphatidylinositol 3-Kinases; Progesterone; Receptors, G-Protein-Coupled; Signal Transduction; Superovulation
PubMed: 27149986
DOI: 10.1530/JOE-15-0521 -
PloS One 2016Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However,...
Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However, the genes encoding NMB and NMBR and their functions especially reproduction of the pigs are currently not fully understood. To research the physiological functions of NMB, we cloned and analyzed the NMB and NMBR genes, and systematically investigated the expression levels of NMB and NMBR mRNA using relative real-time PCR and the distribution of NMBR by immunohistochemistry (IHC). Experimental results show that the sequences of the amino acid and gene of NMB and NMBR were highly conservative and homology in many species, Significantly, the relative RT-PCR results revealed that NMB was mainly expressed in the central nervous system (CNS), whereas NMBR is highly expressed in peripheral tissues and organs, such as endocrine tissues, glands and reproductive organs. The IHC results show that NMBR positive cells were widely distributed in the body, such as respiratory and circulatory system, digestive system, urogenital system, in lymphatic organs and in the endocrine system. We also systematically investigated expression levels of NMB and NMBR in the reproductive axis using relative real-time PCR. In sow estrous cycle, the hypothalamic levels of both NMB and NMBR mRAN were similar, but the expression levels of the pituitary were negatively correlated. Expression levels in the ovarian system are lowest in metestrus phases and highest in proestrus and estrus phases. In boar post-natal development stages, the hypothalamic, pituitary and testicular levels of NMB and NMBR mRNAs showed developmental changes on postnatal day 30, 60, 90 and 120. Taken together, this study provided molecular and morphological data necessary for further research of physiological function of NMB/NMBR system in the pigs.
Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Female; Gene Expression; Male; Molecular Sequence Data; Neurokinin B; RNA, Messenger; Receptors, Bombesin; Reproduction; Swine
PubMed: 27010315
DOI: 10.1371/journal.pone.0151871 -
Veterinary Medicine International 2016The aim of this study was to investigate the period of estrus cycle in aceh cattle, Indonesia, based on vaginal cytology techniques. Four healthy females of aceh cattle...
The aim of this study was to investigate the period of estrus cycle in aceh cattle, Indonesia, based on vaginal cytology techniques. Four healthy females of aceh cattle with average weight of 250-300 kg, age of 5-7 years, and body condition score of 3-4 were used. All cattle were subjected to ultrasonography analysis for the occurrence of corpus luteum before being synchronized using intramuscular injections of PGF2 alpha 25 mg. A vaginal swab was collected from aceh cattle, stained with Giemsa 10%, and observed microscopically. Period of estrus cycle was predicted from day 1 to day 24 after estrus synchronization was confirmed using ultrasonography analysis at the same day. The result showed that parabasal, intermediary, and superficial epithelium were found in the vaginal swabs collected from proestrus, metestrus, and diestrus aceh cattle. Proportions of these cells in the particular period of estrus cycle were 36.22, 32.62, and 31.16 (proestrus); 21.33, 32.58, and 46.09 (estrus); 40.75, 37.58, and 21.67 (metestrus); and 41.07, 37.38, and 21.67 (diestrus), respectively. In conclusion, dominant proportion of superficial cell that occurred in estrus period might be used as the base for determining optimal time for insemination.
PubMed: 26977335
DOI: 10.1155/2016/3976125 -
Genetics and Molecular Research : GMR Jan 2016Frizzled 3 is an important receptor in the Wnt/β-catenin pathway, a conserved signaling pathway that regulates gene expression and controls diverse developmental...
Frizzled 3 is an important receptor in the Wnt/β-catenin pathway, a conserved signaling pathway that regulates gene expression and controls diverse developmental processes. However, the role of this protein during follicular development in the adult ovary is not known. The present study was designed to investigate the expression and localization of Frizzled 3 mRNA and protein during the estrous cycle in the mouse ovary through in situ hybridization (ISH), real-time quantitative polymerase chain reaction, immunohistochemistry and western blot. ISH results showed that in proestrus, high expression of Frizzled 3 was found in the granulosa and stroma with weak levels in the corpus luteum. In estrus and diestrus, the stroma had high Frizzled 3 expression, but levels were low in granulosa cells and corpus luteum. In the metestrus, moderate expression of Frizzled 3 was found in the stroma but low to no expression was found in luteal cells and follicles. The mRNA and protein levels of Frizzled 3 were found to be the highest in proestrus and diestrus compared to estrus and metestrus (P < 0.05), confirming the ISH results. During estrus and diestrus, high Frizzled 3 expression was observed in the stroma and moderate levels in granulosa cells, and during estrus and proestrus, low expression was seen in the oocyte cell membrane. The western blot results further confirmed this change during the estrous cycle. Together, these results indicate that Frizzled 3 is involved in regulating follicular development and oocyte maturation during the estrous cycle.
Topics: Animals; Corpus Luteum; Diestrus; Estrus; Female; Frizzled Receptors; Gene Expression Regulation, Developmental; In Situ Hybridization; Mice; Mice, Inbred ICR; Oocytes; Ovarian Follicle; Proestrus; Real-Time Polymerase Chain Reaction; Time Factors
PubMed: 26909900
DOI: 10.4238/gmr.15017152