Vitrification and transfer of cynomolgus monkey (Macaca fascicularis) embryos fertilized by intracytoplasmic sperm injection.

Protocols for cryopreservation of monkey embryos are not well established. The objective of the current study was to determine the efficacy of the polypropylene strip method for cryopreserving cynomolgus monkey embryos. Cynomolgus monkey embryos, 63 and 56 at the 4- to 8-cell and 56 blastocyst stages, respectively, were produced by intracytoplasmic sperm injection and in vitro culture, and vitrified using a polypropylene strip. For these two stages, 95 and 86% survived after thawing and pregnancy rates were 29.2% (7 pregnant females/24 recipients, with three live births) and 0% (n = 16 recipients). These were apparently the first live births obtained from embryos fertilized by ICSI. In conclusion, 4- to 8-cell preimplantation cynomolgus monkey embryos were successfully cryopreserved using a polypropylene strip.

Canine oocyte maturation in culture: significance of estrogen and EGF receptor gene expression in cumulus cells.

We examined the role of cumulus cells regarding in vitro maturation of canine oocytes, and investigated estrogen and epidermal growth factor (EGF) receptor gene expression and action on nuclear maturation. Canine cumulus-oocyte complexes (COC) were collected from anestrous and diestrous bitches; only COC with vitelline diameter >100 microm were used. In Experiment 1, expression of estrogen receptor (ER) alpha, ERbeta and EGF-receptor (EGF-R) were determined by reverse transcription-polymerase chain reaction (RT-PCR), using mRNA from the oocyte or cumulus cell. Transcripts for the ERbeta and EGF-R were detected in oocytes and cumulus cells, but no message was detected for ERalpha. In Experiment 2, intact COC and the denuded oocytes were cultured in TCM199 medium supplemented with various concentrations of estradiol-17beta (E(2); 0-10 microg/mL) or EGF (0-100 ng/mL) for 72 h; nuclear maturation was then evaluated. In oocytes cultured within intact COC, the rate of germinal vesicle breakdown (GVBD) was higher in the 1 microg/mL E(2) supplemented group (P<0.05), and the rate of metaphase I (MI) was higher in the 10 ng/mL EGF supplemented group, than in the non-supplemented group (P<0.05). However, supplementation of E(2) or EGF to denuded oocytes failed to promote nuclear maturation. In Experiment 3, intact COC were cultured in TCM199 supplemented with 1 microg/mL E(2), 10 ng/mL EGF, and 10% fetal bovine serum (FBS) for 72 h, and nuclear maturation was evaluated. There was no significant difference in the rate of metaphase II (MII) between the medium only, E(2)+EGF, and FBS supplement groups. When E(2) and EGF in combination with FBS were supplemented, the rate of MII was higher than in other groups (P<0.05). We inferred that cumulus cells were involved in mediating the stimulatory effects of E(2) and EGF on nuclear maturation of canine oocytes, and that E(2) and EGF in combination with FBS promoted the completion of oocyte meiotic maturation.

Membrane localization of protein-tyrosine phosphatase 1B is essential for its activation of sterol regulatory element-binding protein-1 gene expression.

Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor in stimulating lipogenesis in the liver. Protein-tyrosine phosphatase 1B (PTP1B) induces SREBP-1 gene expression via protein phosphatase 2A (PP2A) activation. PTP1B is reported to be anchored on the endoplasmic reticulum (ER) via its C-terminal tail, and change in intracellular localization of PTP1B by C-terminal-truncation did not alter its inhibitory effects on insulin signaling. In this study, we investigated whether the change in intracellular localization of PTP1B could influence SREBP-1 gene expression. Overexpression of C-terminal truncated PTP1B (PTP1BdeltaCT) in rat Fao cells did not induce SREBP-1 gene expression. Furthermore, PTP1BdeltaCT failed to bind PP2A, resulting in impaired PP2A activation, whereas overexpression of wild-type PTP1B (PTP1BWT) associated with PP2A. Moreover, a membrane-targeted PTP1BDeltaCT activated PP2A with restored PP2A binding, despite the absence of its C-terminal region. Finally, overexpression of PTP1BdeltaCT into mouse primary cultured hepatocytes failed to enhance SREBP-1c mRNA, whereas membrane-targeted PTP1BdeltaCT led to enhanced SREBP-1c mRNA in hepatocytes as well as PTP1BWT. In conclusion, membrane localization of PTP1B is essential for PP2A activation, which is crucial for its enhancement of SREBP-1 gene expression.

Reduction of mucin-1 gene expression associated with increased Escherichia coli adherence in the canine uterus in the early stage of dioestrus.

The relation between adherence of Escherichia coli and expression of mucin-1 (Muc1: an integral membrane mucin) mRNA in the endometrium was studied in beagle bitches at different stages of the oestrous cycle and in those with cystic endometrial hyperplasia/pyometra complex (pyometra). The number of E. coli adhering to the endometrium was low at pro-oestrus and oestrus and increased at the early stage (day 10) of dioestrus, corresponding to the implantation period; it declined thereafter. Adhesion of the organisms to endometrial epithelial cells collected at day 10 of dioestrus was inhibited by the addition of D-mannose. When endometrial epithelial cells collected at pro-oestrus were treated with hyaluronidase, an enzyme that digests mucins, the numbers of E. coli adhering to the cells tended to increase. With polymerase chain reaction analysis it was possible to detect Muc1 gene transcripts in the endometrium at all stages of the oestrous cycle, although the level of Muc1 mRNA decreased by day 10 of dioestrus. The levels of Muc1 mRNA in bitches with a clinical stage of pyometra were low and comparable to those at day 10 of dioestrus. The number of E. coli adhering to the endometrium and Muc1 mRNA levels in the endometrium were inversely correlated (r=-0.77, P<0.01). Immunohistochemical analysis showed little staining for Muc1 in the endometrial epithelia at day 10 of dioestrus and in bitches with pyometra. These results suggest that reduction of Muc1 expression is associated with increased E. coli adherence in the canine uterus at the early stage of dioestrus, possibly facilitating the development of pyometra.

Stable maintenance of monkey embryonic stem cells in the absence of bFGF.

Monkey embryonic stem (ES) cells are useful tools in preclinical studies of gene therapy and tissue engineering as well as in primate developmental biology. However, their maintenance is not easy, requiring addition of bFGF to the medium. Herein, we have described a stable, cost-effective method that does not require bFGF. We used a high-density (1 to 1.5x10(5) cells/cm2) of mouse embryonic fibroblasts (MEF) as feeder cells to successfully maintain undifferentiated monkey ES cells for 2 years (approximately 150 passages). Furthermore, these ES cells were competent for electroporation of enhanced green fluorescent protein (EGFP) and subsequent drug selection procedures. We were able to establish EGFP-expressing cell lines using this culture condition. These cell lines expressed undifferentiated markers, such as alkaline phosphatase, SSEA-4, TRA-60, and TRA-81. In addition, strong EGFP expression was observed after differentiation into cardiomyocytes, neurons, or adipocytes, suggesting that these cell lines are a useful tool to study cell transplantation. This method simplifies the culture of monkey ES cells.

Lactoferrin expression in the canine uterus during the estrous cycle and with pyometra.

The expression of lactoferrin, a non-specific antimicrobial defence, in the canine uterus during the normal estrous cycle and in bitches with pyometra was examined. Using polymerase chain reaction analysis, lactoferrin gene transcripts were detected in the endometrium at all stages of the estrous cycle, with the highest levels in estrus. In normal bitches, endometrial lactoferrin mRNA increased from proestrus to estrus (P<0.05). Thereafter, it dramatically decreased from estrus to Day 10 of diestrus (P<0.05), and stayed low at Day 35 of diestrus and anestrus; this was consistent with blood estrogen concentrations. Levels of lactoferrin mRNA were higher in bitches with pyometra than in normal diestrus (P<0.05). With immunohistochemistry, distinct staining of lactoferrin was detected in the luminal and glandular epithelial cells of the endometrium at proestrus and estrus, but little staining was detected at Day 10 of diestrus. At Day 35 of diestrus and anestrus, a partial and weak reaction was present in the same region. In bitches with pyometra, the glandular epithelial cells and many cells in the uterine stroma were strongly stained. Staining cells in the stroma were morphologically similar to neutrophils. No lactoferrin staining was seen in the uterine stromal cells or myometrium in any section. These results suggest that, in the canine uterus, lactoferrin expression is related to the blood concentration of estrogen, and that the dramatic reduction in lactoferrin observed at the early stage of diestrus may impair antimicrobial defense. Also, enhanced expression of lactoferrin mRNA in the endometrium with pyometra may be associated with neutrophil invasion into the uterus to combat the infection.

Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI.

Human embryonic stem (ES) cells are predicted to be a valuable source for producing ES-derived therapeutic spare tissues to treat diseases by controlling their growth and differentiation. To understand the regulative mechanisms of their differentiation in vivo and in vitro, ES cells derived from nonhuman primates could be a powerful tool. We established four ES cell lines from cynomolgus monkey (Macaca fascicularis) blastocysts produced by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The ES cells were characterized by the expression of specific markers such as alkaline phosphatase and stage-specific embryonic antigen-4. They were successfully maintained in an undifferentiated state and with a normal karyotype even after more than 6 months of culture. Pluripotential competence was confirmed by the formation of teratomas containing ectoderm-, mesoderm-, and endoderm- derivatives after subcutaneous injection into SCID mice. Differentiation to a variety of tissues was identified by immunohistochemical analyses using tissue-specific antibodies. Therefore, we established pluripotent ES cell lines derived from monkeys that are widely used as experimental animals. These lines could be a useful resource for preclinical stem cell research, including allogenic transplantation into monkey models of disease.

New hyperprolactinemia and anovulation model in common marmoset (Callithrix jacchus) and effect of cabergoline.

We aimed to develop an anovulation model, using sulpiride-induced hyperprolactinemia in common marmosets. The serum prolactin level gradually increased during the twice-daily administration of sulpiride and reached a plateau after 4 days. Sulpiride produced as big a response at 10 mg kg(-1) as at 50 mg kg(-1). In this study, the length of the ovarian cycle was approximately 30 days in normal common marmosets. Serum progesterone and estradiol levels showed no consistent change during the first 2 months of treatment with sulpiride. When treatment with sulpiride had been continued for more than 2 months, serum progesterone and estradiol levels fell to within the range seen in the follicular phase of the normal cycle and absence of ovulation was recognized by laparoscopy. A single oral administration of cabergoline (at doses between 0.01 and 0.1 mg kg(-1)) dose dependently reduced the elevated serum prolactin level. Bromocriptine (at an oral dose of 10 mg kg(-1)) also reduced the serum prolactin level at 4 and 8 h after its administration. With bromocriptine, the prolactin level had recovered at 24 h, but with cabergoline at doses of 0.05 mg kg(-1) or more, it had still not recovered at 48 h. In anovulatory common marmosets, oral administration of cabergoline at a daily dose of 0.05 mg kg(-1) restored ovarian function and resulted in ovulation in 100% of the group (following a reduction in the serum prolactin level). Bromocriptine at a daily oral dose of 10 mg kg(-1) resulted in ovulation in 67% of the group, but this dose was about 200 times higher than the dose of cabergoline. We could produce an anovulatory model induced by sulpiride repeatedly administered over a long time period. It is suggested that, in this anovulatory model in common marmosets, cabergoline has a potent and long-lasting action as a dopamine D2 receptor agonist, and thus could be a useful drug for the treatment of galactorrhea and hyperprolactinemic amenorrhea and/or anovulation.

Increased LH pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch.

The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up-regulated by estradiol.

Induction of fertile estrus in bitches using a sustained-release formulation of a GnRH agonist (leuprolide acetate).

A single subcutaneous injection of a sustained-release formulation of a potent GnRH agonist, leuprolide acetate (LA; [D-Leu6, Pro9NEt]-GnRH), was evaluated as a method of inducing fertile estrus in 12 mature anestrous and 6 prepubertal beagle bitches. The bitches were treated with microencapsulated LA (100 micrograms/kg, s.c.) at 120 or 150 d post partum, or at 1 yr of age, followed by a GnRH-analogue (fertirelin; [Pro9NEt]-GnRH, 3 micrograms/kg, i.m.) on the first day of induced estrus. Signs of estrus were seen within 10.3 +/- 0.9 d after LA administration in all bitches. The interestrous interval in 120- and 150-d post-partum bitches was shortened (P < 0.05) to 191 +/- 3 and 222 +/- 3 d, respectively, compared with 264 +/- 11 d in control bitches. All LA treated dogs demonstrated behavioral estrus and mated. Three of 6 (50%) at 120 d post partum, 6 of 6 (100%) at 150 d post partum and 5 of 6 (83%) of prepubertal (1-yr old) bitches then became pregnant and produced a mean litter size of 4.1 +/- 0.8 pups. A normal circulating estrogen and progesterone response pattern was observed in mature anestrous bitches. A prepubertal bitch that failed to become pregnant had a similar estrogen response pattern but an insufficient progesterone profile. The results suggest that microencapsulated LA can be useful in inducing fertile estrus in the domestic dogs.

Enhancement of estrogen receptor gene expression in the mediobasal hypothalamus during anestrus in the beagle bitch.

Estrogen receptor mRNA (ER mRNA) levels were measured in the mediobasal hypothalamus (MBH) of beagle bitches at different stages of the estrous cycle, and compared with levels in ovariectomized (OVX) estrogen-treated bitches. In cyclic bitches, the level of hypothalamic ER mRNA increased during the progression of anestrus and declined thereafter. Hypothalamic ER mRNA and plasma luteinizing hormone (LH) levels during anestrus and proestrus were positively correlated (r = 0.94, P < 0.001). In OVX bitches, levels of hypothalamic ER mRNA were low, and increased significantly after treatment with a low dose of estradiol benzoate. These results suggest that, during the course of anestrus in the bitch, hypothalamic ER mRNA expression increases, and may be up-regulated by estradiol.

Increasing gonadotropin-releasing hormone release by perifused hypothalamus from early to late anestrus in the beagle bitch.

An in vitro perifusion system was used to investigate pulsatile gonadotropin-releasing hormone (GnRH) release from hypothalamic fragments derived from beagle bitches at different stages of the estrous cycle. The spontaneous GnRH release from the excised tissue fragments that include the "mediobasal hypothalamic-preoptic area-suprachiasmatic nucleus units' was episodic throughout all stages of the estrous cycle with a significantly high release rate during late anestrus and late proestrus. The GnRH release rate and plasma levels of luteinizing hormone were positively correlated (r = 0.94, P < 0.01). These results suggest that during the course of anestrus in the bitch the GnRH release rate increases while the pituitary responds accordingly.

Enzyme immunoassay of gonadotropin releasing hormone in the canine hypothalamus and plasma using monoclonal antibodies.

A monoclonal antibody against gonadotropin releasing hormone (GnRH)-BSA was used in the development of a sensitive enzyme immunoassay of GnRH in the canine hypothalamus and in plasma. The method had a limit of detection of 4 pg per sample. The intra- and interassay coefficients of variation were < 7.3% and < 11.0%, respectively. Acid extracts of hypothalamus produced a dose response curve which was parallel to that obtained with the synthetic GnRH standard. Checking cross reactivity of various fragments of GnRH revealed that the antibody was formed predominantly against the C-terminal end of GnRH. Thyrotropin-releasing hormone and other hypothalamic hormones did not appear to influence the assay. In male dogs, hypothalamic GnRH levels increased with age up to 4 months, then fell to a plateau from 6 months to 2 years. The time required for a 50% reduction in plasma levels following intravenous administration of synthetic GnRH to five adult male dogs was 2.2 +/- 0.1 (SEM) min.

[Hypothalamo-pituitary-ovarian function in female Japanese monkeys (Macaca fuscata) in the non-mating season].

We investigated the mechanism of reduction of hypothalamo-pituitary-ovarian function in female Japanese monkeys in the non-mating season. We administered PMSG and LH-RH to 19 females in the non-mating and in the mating season. When PMSG was administered every day for 12 to 14 days to seven monkeys in the non-mating season, follicle development was observed together with an increase in serum estradiol-17 beta (E2), but there was no rise in serum LH in two animals and little increase in five others. Follicle involution began about ten days after PMSG administration, and no ovulation occurred. These findings show that secretion of LH by the pituitary in response to positive feedback by the E2 secreted by the follicles which had developed was in adequate to induce ovulation. Serum LH levels increased markedly in five females after a single iv injection of LH-RH in the mating season, but not at all in the non-mating season, even when five-times the dose was administered. These data show that the LH-secreting function of the pituitary is definitely reduced in the non-mating season. When LH-RH was administered to seven monkeys following PMSG administration, an LH surge was observed in all animals, and ovulation occurred in four animals. These findings suggest that one reason for the reduction in pituitary-ovarian function in the non-mating season was a decline in LH-RH secretion by the hypothalamus.

[Periovulatory time courses of serum LH in the Japanese monkey (Macaca fuscata)].

Serum LH, E2-17 beta and progesterone concentration were measured in 16 cycles of 15 female Japanese monkeys. Three of the 16 cycles were ascertained to be anovulatory. Ten of the 13 ovulatory cycles showed LH peaks varying from 25 to 280 ng/ml. However, in remaining 3 cycles, LH peak could not be determined, probably because of a lag of blood-sampling schedule. E2-17 beta peaks were detected 0-30 hrs before LH peak in 8 cycles, but 13 or 20 hrs after LH peak in 2 cycles. Time-intervals from LH peak to ovulation ranged 0-47 hrs 30 min. No correlation was detected between concentrations of LH and progesterone in the luteal phase.