A Sexually Dimorphic Area of the Dorsal Hypothalamus in Mice and Common Marmosets.

We found a novel sexually dimorphic area (SDA) in the dorsal hypothalamus (DH) of mice. The SDA-DH was sandwiched between 2 known male-biased sexually dimorphic nuclei, the principal nucleus of the bed nucleus of the stria terminalis and the calbindin-sexually dimorphic nucleus, and exhibited a female-biased sex difference in neuronal cell density. The density of neurons in the SDA-DH was increased in male mice by orchidectomy on the day of birth and decreased in female mice by treatment with testosterone, dihydrotestosterone, or estradiol within 5 days after birth. These findings indicate that the SDA-DH is defeminized under the influence of testicular testosterone, which acts via both directly by binding to the androgen receptor, and indirectly by binding to the estrogen receptor after aromatization. We measured the activity of SDA-DH neurons with c-Fos, a neuronal activity marker, in female mice during maternal and sexual behaviors. The number of c-Fos-expressing neurons in the SDA-DH of female mice was negatively correlated with maternal behavior performance. However, the number of c-Fos-expressing neurons did not change during female sexual behavior. These findings suggest that the SDA-DH contains a neuronal cell population, the activity of which decreases in females exhibiting higher performance of maternal behavior, but it may contribute less to female sexual behavior. Additionally, we examined the brain of common marmosets and found an area that appears to be homologous with the mouse SDA-DH. The sexually dimorphic structure identified in this study is not specific to mice and may be found in other species.

Molecular Cloning of Ghrelin and Characteristics of Ghrelin-Producing Cells in the Gastrointestinal Tract of the Common Marmoset (Callithrix jacchus).

Ghrelin was first isolated from human and rat as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In the present study, we determined the ghrelin cDNA sequence of the common marmoset (Callithrix jacchus), a small-bodied New World monkey, and investigated the distribution of ghrelin-producing cells in the gastrointestinal tract and localization profiles with somatostatin-producing cells. The marmoset ghrelin cDNA coding region was 354 base pairs, and showed high homology to that in human, rhesus monkey, and mouse. Marmoset ghrelin consists of 28 amino acids, and the N-terminal region is highly conserved as found in other mammalian species. Marmoset preproghrelin and mature ghrelin have 86.3% and 92.9% homology, respectively, to their human counterparts. Quantitative RT-PCR analysis showed that marmoset ghrelin mRNA is highly expressed in the stomach, but it is not detected in other tissues of the gastrointestinal tract. In addition, a large number of ghrelin mRNA-expressing cells and ghrelin-immunopositive cells were detected in the mucosal layer of the stomach, but not in the myenteric plexus. Moreover, all the ghrelin cells examined in the stomach were observed to be closed-type. Double staining showed that somatostatin-immunopositive cells were not co-localized with ghrelin-producing cells; however, a subset of somatostatin-immunopositive cells is directly adjacent to ghrelin-immunopositive cells. These findings suggest that the distribution of ghrelin cells in marmoset differs from that in rodents, and thus the marmoset may be a more useful model for the translational study of ghrelin in primates. In conclusion, we have clarified the expression and cell distribution of ghrelin in marmoset, which may represent a useful model in translational study.

Delay of ovulation due to diets containing levonorgestrel in cynomolgus monkeys (Macaca fascicularis).

This study was undertaken to develop a simple and practical method to control the time of ovulation in cynomolgus monkeys. Diets containing a synthetic gestagen, levonorgestrel (LNG) were given daily to normally cycling female monkeys for 2 weeks, and plasma concentrations of estradiol-17ß and progesterone were determined by EIA in order to estimate the time of ovulation. Doses of LNG (0, 3.2, 8, 20, 50, or 125 µg) were given from Day 2 (Day 0 =the first day of menstruation) through Day 15. The numbers of days from the last administration of LNG to the estimated ovulation in the groups treated with LNG at 20 µg and above were significantly greater than those in the controls, and the values in the group treated with LNG at 50 µg were within a narrow range. In a second experiment, LNG was administered at 50 µg in different phases of the menstrual cycle (Days 9-22, 16-29, and 23-36), and the results indicated that ovulation occurred more than 12 days after the last administration in all monkeys, and the number of days from the last administration of LNG to the estimated ovulation in the group treated on Days 16-29 (luteal phase) was significantly greater than that in the group treated on Days 23-36. These results indicate that daily provision of a diet containing 50 µg LNG could be applicable for delaying ovulation, and suggest that the total level of (exogenous and endogenous) progestins is critical for determining the length of ovulation delay in cynomolgus monkeys.

Factors associated with patency of the uterine cervix in bitches with pyometra.

This study examined factors involved in the patency of uterine cervices in the bitch with pyometra. The uterine cervices were obtained from the bitches with pyometra at the time of ovariohysterectomy. Cervical patency was measured by inserting the stainless steel rods with different diameter into cervical canals. Collagen concentration and collagenase activity (for type I collagen) in the tissue were determined and the number of neutrophils, which contain the enzymes related to collagen metabolism, and morphological changes in collagenous fibers were studied by histological examination. Levels of mRNA expressions for hormonal factors, estrogen receptor-α (ER-α), progesterone receptor (PR), relaxin (Rlx) and an attractant of neutrophils, interleukin-8 (IL-8), were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). In the statistical analysis, the cervical patency positively correlated with the collagenase activity, and negative correlation was found between the cervical patency and collagen concentration. Histological examination indicated distinct positive correlation between the cervical patency and the number of neutrophils in the cervical stroma and that the collagenous fiber in the uterine cervix became thinner and degraded with increase of the cervical patency. Although there was no relationship between the cervical patency and the level of mRNA for ER-α, PR or Rlx, IL-8 mRNA level has significant positive correlation with the cervical patency and the number of neutrophils in the cervical stroma. These results suggest that the increased number of neutrophils in the uterine cervix, which could be related to the local expression of IL-8, may be involved in collagen degradation and connective tissue remodeling to increase cervical patency in the bitch with pyometra.

Detection of relaxin mRNA in the corpus luteum, uterus, and uterine cervix in the bitch.

In the pregnant bitch, the placenta is a major source of circulating relaxin, but its local expression in the reproductive organs is not clear. This study demonstrated expression of relaxin mRNA in the corpus luteum, uterus, uterine cervix as well as placenta in the pregnant and nonpregnant bitch by reverse transcriptase-polymerase chain reaction (RT-PCR).