Progranulin (PGRN) is a growth modulating factor released by a variety of cells. This molecule has gained the attention of the neuroscience community with recent discoveries of multifunctional roles of PGRN in normal brain and neurodegenerative disorders. We focus on novel roles of PGRN as a sex steroid-responsible gene in the developing and adult rodent brain. While the developing brain is feminine by default, hormone exposure, including androgen and estrogen, induces masculinization during the critical period. We have shown that PGRN is a sex steroid-responsible gene that may be involved in masculinization of the perinatal rat brain. We also found that in adult rats PGRN gene expression was up-regulated by estrogen in the hippocampus, suggesting that PGRN may mediate the mitogenic effects of estrogen in the active area of neurogenesis. Since it has been recently reported that mutations in PGRN gene are responsible for a type of frontotemporal lobar degeneration in humans, PGRN appears to be also involved in modulating neurodegeneration. Together, PGRN gene expression is induced by estrogen in both developing and adult brains, and it may play multifunctional roles in the organization of functional masculinization in the developing brain and the maintenance of adult brain function.

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Epigenetic reprogramming in early preimplantation embryos, that refers to erasing and remodeling epigenetic marks such as DNA methylation, is essential for differentiation and development. In many species, paternal genome is subjected to genome-wide active demethylation before the DNA replication commences, while maternal genome maintains its methylation status until being demethylated passively during the subsequent cleavage divisions. The purpose of this manuscript was to review the available knowledge about the paternal genome active demethylation process concerning the possible mechanisms, species variation and the factors affecting the active demethylation dynamics such as in vitro protocols for production of pronuclear-stage zygotes. Better understanding the mechanisms by which the epigenetic reprogramming is occurred may contribute to clarify the biological significance of this process.

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POU5F1 and NANOG play important roles in the maintenance of embryonic stem cell pluripotency. Recently, we isolated cat embryonic stem (ES)-like cells from cat blastocysts generated in vivo. In an effort to identify genetic markers for the characterization of cat ES-like cells, we have determined the coding sequences (CDSs) of cat POU5F1 (cPOU5F1) and NANOG (cNANOG). The sequence identities of cPOU5F1 with orthologous genes of the human and mouse were 92 and 82%, respectively, at the nucleotide level and 94 and 83%, respectively, at the amino acid level. We identified POU-specific and POU homeodomain sequences in the CDS of cPOU5F1. The sequence identities of cNANOG with its human and mouse orthologs were 69 and 68%, respectively, at the nucleotide level and 69 and 58%, respectively, at the amino acid level. We identified a homeodomain, SMAD4 domain and tryptophan repeat domain (W/QXXXX) in the CDS of cNANOG. We examined the expression of cPOU5F1 and cNANOG mRNA in ES-like cells and fibroblast feeder cells by RT-PCR. Transcripts of cPOU5F1 and cNANOG were detected at a high level in ES-like cells. However, these two genes were undetectable in cat fibroblast feeder cells and 6 adult tissues. We also examined ES-like cells by immunocytochemistry and demonstrated that cPOU5F1 and cNANOG are present at high levels in cat ES-like cells and are undetectable in cat fibroblast feeder cells. These results confirmed that cat ES-like cells can be successfully isolated from in vivo-produced blastocysts and that the expression of cPOU5F1 and cNANOG can be used as a biomarker for characterization of cat ES-like cells.

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Vanadate, an inhibitor of tyrosine phosphatases, has been reported to prevent germinal vesicle breakdown in mammalian oocytes. We examined the effect of vanadate on the chromatin configuration of fully grown pig oocytes. In the presence of human menopausal gonadotropin (hMG), vanadate (0.5-5 mM) resulted in a dose-dependent change in oocyte chromatin in germinal vesicles from the condensed state to a decondensed filamentous or stringy configuration. The effect of vanadate and hMG on chromatin configuration could be replicated with 2 mM dibutyryl cyclic AMP (dbcAMP) in place of hMG. Western blot analysis showed that vanadate caused a massive accumulation in the oocytes of tyrosine-phosphorylated proteins with a range of molecular weights that was enhanced by both hMG and dbcAMP in a similar manner. These results suggest that inhibition of tyrosine phosphatase(s) in the presence of an effective level of cAMP induces a change in chromatin configuration of pig oocytes.

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The aims of this study were to investigate improvements to the pig preimplantation embryo culture system using in vitro produced embryos. For experiment 1, the optimum time to change the medium from NCSU23 containing 0.6 mM glucose, 0.2 mM pyruvate, 5.7 mM lactate and nonessential amino acids to NCSU23 containing 5.6 mM glucose and both essential and nonessential amino acids was examined. There were no statistically significant differences in blastocyst rates or cell number when the medium was changed at 48, 72 or 96 h, although there was a consistent trend for the 96 h treatment to produce fewer blastocysts with fewer cells. For experiment 2, the addition of essential amino acids at either a 1:50 or a 1:100 dilution of the purchased stock solution for day 1 to 6 or for days 3 to 6 only was investigated. Adding essential amino acids at a 1:50 dilution for day 3 to 6 significantly reduced the blastocyst rate and adding them at a 1:50 dilution from day 1 to 6 significantly reduced both the blastocyst rate and blastocyst cell number compared to when it was added at a 1:100 dilution. Embryos were produced by IVF, cultured for 6 days and good quality blastocysts were transferred into 6 synchronized pseudopregnant recipients (24 to 35 blastocysts per recipient) resulting in 4 pregnancies and 21 live birth piglets. These results show that adding essential amino acids at a 1:100 dilution provided the best culture conditions and the blastocysts produced were able to attain full term development after transfer.

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The aim of the present study was to compare the effects of full-length rat kisspeptin (rKp-52) with C-terminal decapeptide (Kp-10) of rat or human kisspeptin on LH release in intact male rats. Plasma LH profiles were determined by frequent blood sampling at 6-min intervals for 3 h after central or peripheral injection of kisspeptins. Intracerebroventricular (icv) injection of rKp-52 (0.1 nmol) induced a gradual increase in the plasma LH level, which remained high for the rest of the sampling period. On the other hand, icv injection of rKp-10 did not increase the plasma LH level at the same dose (0.1 nmol). A 10-times higher dose (1 nmol) of rKp-10 and hKp-10 increased the plasma LH level, but the increase was lower than that of rKp-52 icv injection. Intravenous (iv) injection of kisspeptins also stimulated LH release at 10 or 100 nmol/kg. In rKp-52 (10 nmol/kg)-treated animals, the plasma LH level reached a peak within 30 min and remained high until 60 min postinjection. The rKp-10- and hKp-10-injected animals showed a more rapid decline in plasma LH level after the peak found at around 30 min after the injections at both middle (10 nmol/kg) and high (100 nmol/kg) doses. The present study indicates that full-length kisspeptin is more effective in stimulating LH release compared with Kp-10 in male rats. The difference in LH-releasing activity may be the result of a difference in degradation of the peptides, but it is still worth determining whether an active domain other than the C-terminal decapeptide is present in full-length kisspeptin.

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Polyploid embryo production is an important technique in generating mice directly from embryonic stem (ES) cells. The present study was designed to assess the effect of different calcium concentrations and electric field intensities on the production of tetraploid embryos with higher developmental potential by electrofusion. Two-cell mouse embryos were electrofused in fusion solution containing different concentrations of calcium ion (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mM). The rates of blastomere fusion, and subsequent cleavage and development of tetraploids to the blastocyst stage were highest when two-cell embryos were electrically stimulated in a fusion medium containing 1.0 mM calcium. Therefore, we tested electric field intensities (0.6, 0.8, 1.0, 1.2 and 1.4 kV/cm) for electrofusion of two-cell embryos and subsequent development to the blastocyst stage in 1.0 mM calcium. The highest rates of fusion and blastocyst formation were observed when the electric field strength was 0.8 kV/cm. The present results showed that mouse two-cell embryos stimulated with 0.8 kV/cm in a fusion medium containing 1.0 mM calcium had the highest rates of fusion and development to the blastocyst stage.

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Cryopreservation of mouse sperm is useful for maintaining various strains. However, fertility generally decreases after freezing. In particular, the fertility of cryopreserved C57BL/6J sperm is very low. To improve the fertility of frozen sperm, we examined the efficiencies of various media used for sperm preincubation (SP) and in vitro fertilization (IVF) in frozen C57BL/6J sperm. In this study, SP medium was examined for efficiency of fertility with respect to content, especially calcium (Ca²⁺), phosphate (PO₄^3-) and lactate. In all media containing no Ca²⁺, including medium lacking Ca²⁺, lacking Ca²⁺ and PO₄^3-, lacking Ca²⁺ and lactate and lacking Ca²⁺, PO₄^3- and lactate, high IVF rates were obtained (79, 69, 76 and 71%, respectively). On the other hand, the rates for media containing Ca²⁺ were significantly lower (30-38%, P<0.05). After transfer, 41-50% of newborns were obtained in all media containing no Ca²⁺. In conclusion, preincubation of thawed sperm in medium containing no Ca²⁺ markedly improved the fertility of cryopreserved C57BL/6J sperm. These results indicate that the present method of IVF using medium with no Ca²⁺ is practical for use in cryopreserved C57BL/6J sperm.

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Lysophosphatidic acid (LPA) is becoming a new player in regulation of the reproductive processes of domestic animals. In the present study, we examined whether LPA modulates prostaglandin (PG) synthesis in the bovine endometrium at the time of the early maternal pregnancy recognition compared with the respective days of the estrous cycle and the enzymatic mechanism of this action. Bovine epithelial and stromal endometrial cells isolated from the uteri on days 8-10 of the estrous cycle and pregnancy were cultured with LPA for 24 h. LPA increased PGE₂ production in stromal cells during the estrous cycle and early pregnancy. On days 8-10 of pregnancy, LPA inhibited PGF_2α production in epithelial cells. LPA stimulated PGES mRNA expression in stromal cells during both examined phases and inhibited PGFS mRNA expression in epithelial cells on days 8-10 of pregnancy. The overall results indicate that LPA may serve as a luteotropic factor during the luteal phase of the estrous cycle and early pregnancy stimulating PGE₂ synthesis and mRNA expression for PGES in stromal cells. Moreover, during early pregnancy, LPA might protect bovine CL and early embryo development by decreasing PGF_2α synthesis and mRNA expression for PGFS in the epithelial cells of the bovine endometrium.

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Endocrine disruptors (EDs) with androgenic and anti-androgenic effects may alter reproductive function by binding to androgenic receptors (AR) and inducing or modulating AR-dependent responses in the male reproductive system. However, the molecular mechanism(s) underlying these events remains unclear. In the present study, pregnant Sprague Dawley (SD) rats were treated with testosterone propionate (TP), flutamide (Flu) and di-(2-ethylhexyl) phthalate (DEHP) from gestation days (GD) 11 to 21. Interestingly, maternal exposure to Flu or DEHP caused fluctuations in the neonatal levels of serum testosterone (T) and luteinizing hormone (LH). Serum testosterone and LH were upregulated by Flu, but these hormones were down-regulated by DEHP. The anogenital distances (AGD) of male newborns were determined at post-neonatal days (PND) 1, 21 and 63. Male rats treated prenatally with DEHP (100 mg/kg mother's body weight) or Flu showed an AGD shorter than that of control rats. At PND 63, sperm concentration, viability and motility were reduced in the maternal DEHP and Flu-treated groups. The numbers of seminiferous tubules were reduced in the Flu and DEHP-treated offspring when compared with the vehicle- and TP-treated groups, and the tubules of the testes at PND 63 were disrupted by a high dose of Flu. In addition, we found differential gene expression patterns by microarray analysis following ED exposure, particularly in sex determination-related genes. Although Flu and DEHP are considered to be identical with regard to their anti-androgenic effects, their effects on developing male reproductive organs were distinct, suggesting that Flu competes with endogenous T, while DEHP influences a different step in androgenesis.

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We aimed to determine plasma progesterone and estradiol-17β concentrations, as well as fecal progesterone concentrations during the estrous cycle in a female 8-year-old Japanese serow (Capricornis crispus). The step frequencies during the night were recorded by a pedometer attached to the serow's hind leg from October to the following June. Estrous behavior was also monitored during the day. Blood samples were taken once a day from 8 February to 10 March from an indwelling catheter placed in the jugular vein. Fecal samples were taken once or twice a day from 1 January to 29 April. Plasma and fecal progesterone and plasma estradiol concentrations were determined using time-resolved fluoroimmunoassays. The estrus behavior observed lasted for 2-3 days. Peak step frequencies were recorded between November and April, at intervals of 17-19 days. Plasma progesterone concentrations remained elevated (1-7.7 ng/ml) for 12 days during non-estrus, and plasma estradiol concentrations were highest when the peak step frequency and estrous behavior were observed. Step frequency increased around the times fecal progesterone levels fell to basal levels. Progesterone concentrations in feces were significantly correlated with those in sera. Thus, measurement of fecal progesterone concentrations might be useful for monitoring the reproductive status of the Japanese serow.

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The objective of the present study was to elucidate whether luteolytic prostaglandin F_2α (PGF) plays roles in regulating the nitric oxide (NO) generating system in luteal endothelial cells (LECs). Reverse transcriptase PCR, immunoblotting and immunostaining revealed the presence of PGF receptor mRNA (521 bp) and protein (64 kDa) in cultured LECs obtained from the mid-stage corpus luteum. When cultured LECs were exposed to 0.1 μM-10 μM PGF, NO production was significantly stimulated by PGF at 24 h. When LECs were exposed to 1 μM PGF for 2, 6 and 24 h, PGF did not affect the expressions of endothelial NO synthase (eNOS) mRNA and protein. On the other hand, PGF stimulated the expression of inducible NOS (iNOS) mRNA (P<0.05) and protein (P<0.05) at 2 h, but not at 6 and 24 h. By observing the conversion of [³C]_L-arginine to [³C]_L-citrulline, we found that PGF stimulated NOS activity in cultured LECs at 2 h (P<0.05). The overall findings indicate that bovine LECs are a target for PGF and that PGF stimulates iNOS expression and NOS activity in bovine LECs. Stimulation of the NO generating system and NOS activity by PGF may result in increasing local NO production followed by luteolysis.

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The aim of this study was to characterize the promoter activity of the porcine pituitary glycoprotein hormone common alpha gene (Cga) promoter (-1059/+12) and the role of LIM homeodomain transcription factor Lhx3. A transfection assay using Chinese hamster ovary (CHO) cells showed that the -1059/-101 region of the Cga promoter definitely responds to Lhx3 and that the -1059/-240 region exhibits a high basal transcriptional level in a pituitary-derived cell line, LβT2. A DNA binding and DNase I footprinting assay demonstrated that Lhx3 has seven binding sites in the -1059/+12 region of Cga, including a pituitary glycoprotein hormone basal element (PGBE) known as a LIM homeodomain factor-binding site. A transfection assay of the sequence of Lhx3-binding sites fused with minimal promoter vector confirmed their Lhx3-dependent stimulations in LβT2 cells. RT-PCR analysis of porcine pituitary ontogeny demonstrated that porcine Lhx3 showed striking changes of expression in both sexes during the fetal period but a stable high level of expression after birth. Thus, the porcine Cga promoter is regulated by Lhx3 through seven sites in the distal and proximal regions.

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This study was designed to investigate the dynamics of the paternal genome demethylation in pronuclear-stage bovine zygotes produced either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) using freeze-thawed (FT) as well as freeze-dried (FD) bull sperm stored at +4 or -196 C for one year. Zygotes were fixed and immunostained using anti-5-methyl-cytosin at 8, 10, 14 and 18 h post IVF (hpi) and at 6 and 12 h post ICSI (hpic). In conventional IVF-derived zygotes, the overall average of the relative methylation (RM; male/female) decreased from 0.92 at 8 hpi to 0.69 at 10 hpi (P<0.05) without any additional decrease at 14 and 18 hpi (0.67 and 0.64, respectively; P>0.05). This was accompanied by higher proportions of zygotes showing RM<0.6 (45.5, 37.5 and 38.2% at 10, 14 and 18 hpi, respectively; P<0.05) compared with 3.7% at 8 hpi. The overall averages of the RM in the FT-ICSI derived zygotes (0.79 and 0.66 at 6 and 12 hpic, respectively) were similar to those in the corresponding IVF-derived zygotes (8 and 14 hpi), but a higher proportion of the 6 hpic zygotes (37.8%; P<0.05) showed an RM<0.6 compared with the 8 hpi zygotes (3.7%). The proportions of FD-ICSI derived zygotes at 12 hpic showing an RM<0.6 (60.6 and 62.4% for +4 and -196 C storage, respectively) were higher than that of the FT-ICSI derived zygotes (39.4%; P<0.05). Thus, the bovine paternal genome rapidly demethylated within 10 h after IVF and 6 h after ICSI, and the freeze-drying and/or the storage process had no adverse effect on demethylation of the paternal genome. The extent of demethylation in the pronuclear-stage bovine zygotes was moderate, with 0.4≤RM<0.6.

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A great deal is known regarding the process of sex differentiation in fish. However, little is known about the presence of immune cells and cytokines in this process. In the gilthead seabream, both immune cells and cytokines play an important role in the tissue reorganization of the gonads during the adult reproductive cycle. We have studied, using light microscopy and immunocytochemistry, the ontogenetic development of the gilthead seabream gonads, focusing on the presence of immune cells and cytokines. We show that the testicular area is quickly differentiated and becomes functional in specimens less than a year old, while the ovarian area differentiates later and continues to develop during the first two years of life. Throughout the morphogenesis process, acidophilic granulocytes were present in the gonad. Interleukin-1b (Il1b) is produced in the testicular area in juveniles and male fish, but not in the ovarian area. Macrophage-colony stimulating factor receptor (Mcsfr) is not produced in the undifferentiated gonad and is only found once the testicular area is well developed.

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To induce meiotic resumption of porcine oocytes, it is thought to be necessary to expose the cumulus-oocyte complexes (COCs) to gonadotropins during in-vitro maturation (IVM). However, the detailed mechanism of meiotic resumption by gonadotropins is still unknown, and successful piglet production has not been reported by using oocytes matured in gonadotropin-free media and fertilized in vitro. The present study was undertaken to examine the combinational effects of epidermal growth factor (EGF)-family members and dibutyryl cyclic AMP (cAMP) in a chemically defined medium on IVM of porcine oocytes and the developmental competence following in vitro fertilization (IVF). The basic IVM medium was a chemically defined medium, modified porcine oocyte medium (mPOM). Supplementation of the IVM medium with 10 or 1000 ng/ml EGF, amphiregulin and betacellulin during the whole IVM period, except for 10 ng/ml amphiregulin, increased the percentage of oocytes maturing to the metaphase-II stage. When COCs were exposed to both dibutyryl cAMP and EGF-family members during the first 20-h of IVM and then culture was continued in the absence of EGF-family members and dibutyryl cAMP, the incidence of metaphase-II oocytes was significantly increased and was not different from that of oocytes cultured in a standard IVM system with gonadotropins. The developmental competence of the oocytes to the blastocyst stage following IVF was no different from that of control oocytes matured with gonadotropins. When these blastocysts were transferred into the uterine horn of three recipients, all of gilts became pregnant and delivered a total of 11 piglets. These observations indicate that supplementation of a chemically defined maturation medium with EGF-family members and dibutyryl cAMP during the first 20 h of IVM can support well the meiotic progress and developmental competence of porcine oocytes.

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This study was undertaken to produce trophoblastic vesicles (TVs) by using blastocysts of in vitro origin and to estimate the effect on the interestrous interval after transfer of 4 TVs into the uteri of heifers on Day 7. Morphological examination under a stereoscopic microscope revealed that the total formation rate of TVs prepared from IVP expanded blastocysts was 80.5% and that there was no difference in the formation rates of TVs derived from blastocysts between Day 7 (83.5%) and Day 8 (78.5%). After intrauterine transfer of TVs, observation of the corpus luteum (CL) by transrectal ultrasonography together with measurement of the plasma progesterone concentration confirmed that 2 of 4 recipients (50%) had a longer interestrous interval, 33.5 and 35.0 days, while the other 2 recipients had normal cycles, 20.0 and 24.5 days. In the control group transferred D-PBS, all 4 heifers had a normal cycles, 24.0-24.5 days. Consequently, the average number of days after intrauterine transfer of TVs compared with the 2 consecutive cycles just before the treatment was longer than in the controls (6.1 ± 2.4 days vs. -0.8 ± 1.1 days, P<0.05). These results indicate that preparation of TVs from blastocysts of in vitro origin is a useable method and that TVs from blastocysts may have the capacity to maintain CL function after intrauterine transfer.

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