Fine tuning of the nervous system in response to intrinsic and extrinsic cues is necessary for successful reproductive behavior. Gonadotropin releasing hormone (GnRH) was originally identified as a hypophysiotropic hormone that facilitates the release of gonadotropins from the pituitary. Although later studies reported their presence, the nonhypophysiotropic GnRH systems, which consist of two groups located in the terminal nerve (TN) and the midbrain tegmentum, respectively, has long been overshadowed by the hypophysiotropic GnRH system. By taking advantage of the teleost brains in which all three GnRH systems are well developed, the anatomical and electrophysiological properties of all three groups of GnRH neurons have been studied. However, despite our increasing endocrinological knowledge, we know very little about the manner of information flow by nonhypophysiotropic neuromodulatory GnRH neurons in the brain. In this article, we will review recent advances in the studies of nonhypophysiotropic GnRH neurons from cellular to behavioral levels. We will first discuss general features of the information processing by peptides and then introduce our recent approaches toward the understanding of the excitation-secretion coupling mechanism of single GnRH neuron using our newly developed primary culture system of isolated TN-GnRH3 neurons. We also introduce autocrine/paracrine regulation of TN-GnRH3 neurons by NPFF peptides for synchronization among them. In addition, we highlight recent advances in the neuromodulatory action of GnRH peptide on the information processing of sensory neuronal circuits and reproductive behavior. These multidisciplinary approaches will greatly advance our understanding of the complex action of GnRH peptides in relation to the brain control of reproduction.

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Unfertilized oocytes are one of the most desired germ cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. The aim of the present study was to improve vitrification of mouse oocytes. First, the effects of calcium and cryoprotectants, dimethyl sulfoxide and ethylene glycol (EG), in vitrification medium on survival and developmental ability of vitrified oocytes were evaluated. Oocytes were vitrified by a minimal volume cooling procedure using different cryoprotectants. Most of the vitrified oocytes were morphologically normal after warming, but their fertility and development were low independently of calcium and cryoprotectants. Second, the effect of cumulus cells on ability of oocytes to be fertilized and develop in vitro was examined. The fertility and developmental ability of denuded oocytes (DOs) after IVF were reduced compared with cumulus-oocyte complexes (COCs) both in fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop to the 2-cell and blastocyst stages than those of vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate equivalent to the rate obtained with IVF using fresh COCs. Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and EG retain their developmental competence. These findings will contribute to improve oocyte vitrification in not only experimental animals but also clinical application for human infertility.

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A chemically defined medium would be useful for analyzing promoters or inhibitors in in vitro culture (IVC) of bovine embryos. However, an IVC system for bovine embryos in a chemically defined medium has not been fully established. The present study was carried out to establish an advanced chemically defined medium for bovine embryos that supports a high rate of embryo development to the blastocyst stage. In the first experiment, we examined the effects of addition of Medium RD (RPMI1640 and Dulbecco's MEM, 1:1 v/v) to mKSOM/aa on developmental competence. The addition of 10% RD to mKSOM/aa with BSA improved the rate of development to the blastocyst stage; however, 10% RD-mKSOM/aa with PVP, which is a chemically defined medium, caused a reduction in the percentage of hatching blastocysts. In the second experiment, embryos were cultured in the chemically defined medium of 10% RD-mKSOM/aa containing 11.7, 23.4, 46.8, 70.2 or 96.8 μM inositol. Inositol at the concentration of 70.2 μM improved the rate of development to the hatching blastocyst stage. In the third experiment, the optimal RD concentration in the IVC medium was evaluated. Embryos were cultured in the chemically defined medium supplemented with 10, 20 or 30% (v/v) RD. The rate of development to the blastocyst stage was highest with 20% RD. In the fourth experiment, the effects of N-acetylglucosamine (GlcNAc) as an IVC medium supplement on developmental competence were examined. The rate of development to the blastocyst stage with 1.0 mM GlcNAc was significantly higher than that without GlcNAc, but the rate of development with 1.2 mM GlcNAc was not different from that without GlcNAc. We also evaluated the ability of blastocysts produced in RD-mKSOM/aa to develop to normal calves after being transferred into recipients. Ten of the 16 recipients became pregnant, with 9 delivering normal calves. These results indicate that 20% RD-mKSOM/aa containing 70.2 μM myo-inositol and 1 mM GlcNAc is useful as a chemically defined medium for IVC of bovine embryos.

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Hatano high- and low-avoidance rats (HAA and LAA strains, respectively) were selected and bred according to the avoidance rate in a shuttle-box task. Although they have clear strain differences in ovarian function, their endocrine mechanisms still remain to be clarified. Differences in female reproductive endocrinology between the strains were investigated by means of measuring the plasma concentration of reproductive hormones during the estrous cycle. LAA rats showed approximately threefold lower basal and surge levels of LH, a more than fourfold lower level of FSH surges and higher levels of inhibin A and inhibin B during the estrous cycle compared with the levels seen in HAA rats. The concentration of estradiol-17β in the proestrous stage was significantly lower in LAA rats than in HAA rats. Additionally, LH and FSH secretions from primary cultured anterior pituitary cells with or without in vitro GnRH stimulation were lower in the cells derived from LAA rats and, in terms of FSH secretion, were unresponsive to GnRH in contrast to cells derived from HAA rats. Although an increased number of preantral follicles in diestrus were observed in LAA rats, number of hCG-induced ovulation was lower in LAA rats. LAA rats may have much more follicle growth during the early stage of folliculogenesis, but most follicles might not grow into mature follicles. These results strongly suggest that the strain difference in ovarian function of these two Hatano rats is due to the difference in the regulation of hypothalamo-hypophyseal system for gonadotropins secretion.

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Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice and plays an important role in other mammals including humans. Intraperitoneal (ip) injections with anti-LIF antibody (7.5 μg/g body weight, 3 times) between D3 (D1 = day of vaginal plug detection) and D4 effectively blocked embryo implantation; complete inhibition was achieved in C57BL/6J mice, and implantation was dramatically reduced in ICR mice (reduced to 27%). Normal rabbit IgG used as the control did not disturb embryo implantation. Anti-LIF antibody was localized not only in the stroma, but also in the luminal epithelium and the glandular lumen after ip injections. Growth-arrested blastocysts were recovered from the uterus without any implantation sites in both strains. Blastocysts made contact with the LE on the antimesometrial side; however, uterine stromal cells did not undergo secondary decidual reaction, and the uterine lumen was open, even at D7. Several regions of decidualization in ICR mice treated with anti-LIF antibody were smaller than those of the control, and development of blastocysts was delayed. The expression of LIF-regulated genes, such as immune-responsive gene-1 and insulin-like growth factor binding protein-3, was significantly decreased in C57BL/6J mice treated with anti-LIF antibody compared with the control, but not in ICR mice. The present study demonstrated that simple ip injections of an antibody are sufficient to block one of the important factors involved in embryo implantation in mice, and this method should also be easily applicable to the investigation of other factors involved in implantation.

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The nutritional deficiencies provoked by ethanol consumption, during gestation or lactation, can contribute to multiple birth defects in offspring. In order to improve our knowledge about selenium (Se) distribution in pups exposed to ethanol, the present study evaluated the effect of this drug on intestinal development and determined its action on duodenal absorption of selenomethionine (Se-Met). To determinate if supplementation could improve Se absorption and its serum values, we used two antioxidant supplemented regimens on dams, with selenium alone or selenium plus folic acid, and obtained six groups of pups: C (control), A (alcohol), CS (control + Se), AS (alcohol + Se), CFS (control + Se + folic acid) and AFS (alcohol + Se + folic acid). Duodenal Se-Met transport was performed using an in vivo perfusion method. Se levels were measured by graphite furnace atomic absorption spectrometry. The supplemented diets utilized had a positive influence on body growth, duodenal perimeter and Se content in ethanol-exposed pups. Ethanol exposure increased Se-Met duodenal absorption in all pups, supplemented or not, presenting the highest values of maximal velocity (V_max) compared with their control counterparts. The affinity constant (K_m) increased according to rank: A>AS>AFS groups. These results suggest that although antioxidant supplementation does not restore Se-Met absorption to normal values, it enhances the affinity of the transporters for the substrate and improves the damage caused by ethanol in the duodenal mucosa.

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This study was undertaken to examine pre- and postimplantation developmental potency of cryopreserved embryos that had undergone in vitro growth (IVG), maturation (IVM) and fertilization (IVF) of oocytes from the preantral follicle stage. An oocyte culture system for IVG and IVM was used in oocyte-granulosa cell complexes (OGCs) derived from preantral follicles in 12-day-old mice. The rate of oocyte maturation was improved by the addition of gonadotropins (FSH / LH) and cytokines (IGF-I / SCF) to culture medium for IVG. During culture for IVG, estradiol-17β and progesterone concentrations increased progressively to the latter period of culture. This culture system enabled IVG, IVM, IVF and pre- and postimplantation development. From 90 cryopreserved 2-cell stage embryos transferred into recipients after warming, 10 live pups were produced. Cryopreservation of embryos by vitrification at the 2-cell stage showed no harmful effect on development to the blastocyst stage or on the cell numbers of the inner cell mass (ICM) and trophectoderm (TE). This study demonstrated that embryos derived from oocytes grown in vitro have tolerance for vitrification and competence to develop to term after warming. This IVG-IVM-IVF technology combined with embryo cryopreservation might be useful for assisted reproduction in mice.

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Since BSE testing of slaughtered cattle is obligatory in Japan, storage of ovaries at 15-20 C overnight in phosphate buffered saline has become a routine protocol in in vitro production (IVP) of cattle embryos. Ovary storage is known to reduce developmental competence of oocytes; however, its effects on oocyte gene expression have not been clarified yet. This study compared oocytes collected from stored slaughterhouse-derived ovaries with those collected by Ovum Pick-Up (OPU) in terms of the expression of 20 selected genes to determine if ovary storage affects cellular processes at the molecular level. Expression of mRNA in oocytes was assayed before and after in vitro maturation (IVM) by real-time quantitative PCR. Maternal mRNA levels of genes were investigated in 2-cell stage embryos obtained from slaughterhouse oocytes to assess their roles for blastocyst formation. In immature OPU oocytes, genes related to metabolism (GAPDH), transporters (GLUT8, ATP1A1) and stress resistance protein (HSP70) showed significantly higher expression compared with oocytes derived from stored ovaries. During IVM, the expression of GDF9, GLUT8, CTNNB1 and PMSB1 was significantly decreased irrespective of oocyte source. Two-cell stage embryos cleaving at 22-25 h after in vitro fertilization (IVF) showed a significantly higher blastocyst formation rate and ATP1A1 gene expression level compared with those cleaving at 27-30 h after IVF. Our results reveal that storage of ovaries alters mRNA levels in oocytes. Correlation of Na/K ATPase ATP1A1expression in IVP embryos at the 2-cell and 8-cell stages with their developmental ability to the blastocyst stage may suggest the importance of maternal mRNA of this gene during blastulation in embryos derived from slaughterhouse oocytes.

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We performed the first genome-wide expression analysis to compare the differences in gene expression in the female sperm reservoir of the duck reproductive tract between two groups with long and short fertile periods to identify factors that may be associated with the fertile period using an oligonucleotide microarray. RNA was extracted from the uterovaginal junction (UV junction) of the two groups. Affymetrix chips containing comprehensive coverage of 32773 transcripts were hybridized with biotin-labeled cRNA, and three biological repeats were performed. We identified 27 transcripts as being differentially regulated. Interestingly, by mapping the differentially expressed transcripts to annotated pathways, we found that Neuropeptide Y (NPY), the RNA expression of which was increased by 2.96-fold in the short-fertile-period group as compared with the long-fertile-period group in our experiment, has been shown to reduce blood flow and substance supply to local tissues. Enah/Vasp-like (EVL), the RNA expression of which was significantly increased by 1.77-fold in the short-fertile-period group as compared with the long-period group, has been demonstrated to be important in activated T-cells. In contrast, trafficking kinesin-binding protein 1 (TRAK1), the expression of which was increased by 2.33-fold in the long-period group as compared with its counterparts, has been suggested to inhibit precocious activation of sperm and prolong sperm life in the female sperm reservoir. The results of real-time PCR confirmed the data obtained by microarray analysis. Our study demonstrated that combining global gene expression investigation with annotated pathway resources contributes to the understanding of sperm life when sustained in the UV junction.

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The objective was to examine the effect of seminal plasma on cryopreservation of sperm from rhesus macaques. Sperm cryosurvival was evaluated by sperm motility and acrosomal integrity. Compared with slow cooling (-0.4 C/min) from 37 C (body temperature) to 4 C, rapid cooling (-16 C/min) caused cold shock in rhesus macaque sperm. The cryosurvival of sperm was decreased regardless of the presence or absence of seminal plasma (P<0.05). However, the presence of seminal plasma during cold shock at a rapid cooling rate improved sperm motility and acrosomal integrity in individual monkeys. Male-to-male variation in sperm cryosurvival was observed after cryopreservation (P<0.05), and the presence of seminal plasma during sperm cryopreservation improved sperm motility and acrosomal integrity in individual monkeys (P<0.05). Furthermore, by adding seminal plasma from monkeys with good sperm cryosurvival to sperm freezing extender, the frozen-thawed motility and acrosomal integrity of sperm from monkey with poor cryosurvival were improved (P<0.05). The present study indicated that seminal fluid is beneficial to sperm undergoing cold shock or cryopreservation in individual monkeys. The cryosurvival of sperm from rhesus macaques with poor sperm freezability could be improved by the presence of seminal plasma from males with good sperm cryosurvival. This finding provides a useful method for genetic preservation in this important species.

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The porcine zona pellucida (ZP) undergoes biochemical changes during the final phase of maturation prior to fertilization. The present study was conducted to elucidate whether the acidification of ZP glycoproteins during porcine oocyte maturation influences sperm-ZP interactions. Two-dimensional gel electrophoresis clearly demonstrated that ZP acidification occurred in accordance with the sialylation and sulfation of ZP glycoproteins in oocytes matured for 44 h. The increases in the incidences of sperm penetration and polyspermy with the progress of the IVM culture period were significantly suppressed by ZP desialylation on treatment with neuraminidase as a consequence of reductions in the number of sperm bound to ZPs and the acrosome reaction (AR) in ZP-bound sperm (P<0.05). In contrast, the blocking of ZP sulfation by NaClO₃ treatment during IVM markedly reduced the incidence of polyspermy with no inhibitory effect on penetration, but the number of sperm bound to ZPs and the rate of AR-inducing sperm were decreased to the same level as in desialylated oocytes. The results indicate that ZP sulfation influences sperm-ZP interactions in a ZP sialylation-independent manner. Moreover, sialylation and sulfation were not associated with a protective proteolytic modification of the ZP matrix before fertilization. These findings suggest that ZP acidification elicited by the sialylation and sulfation of ZP glycoproteins during oocyte maturation contributes to the porcine ZP acquiring the capacity to accept sperm.

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