The effect of estrogen on phosphorylation of prolactin in the mouse pituitary gland

Several studies have indicated that prolactin (PRL) assumes oligomeric, proteolytically cleaved, phosphorylated and glycosylated forms. Phosphorylated PRL (PPRL) is considered to be the most important posttranslationally modified form in the rat. In the present study, we examined whether or not PRL is present in the mouse pituitary gland in the phosphorylated form. Mouse pituitary PRL was digested with acid phosphatase, resolved by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue, and then immunoblotted against the anti-PRL, anti-phosphoserine and anti-phosphothreonine antibodies. We also examined whether PRL is phosphorylated by protein kinases and semi-quantified the ratios of PPRL to PRL in the pituitary gland. The results indicated that three types of PRL are present in the pituitary glands of both male and female mice. One was non-phosphorylated (isoform 1), and the other two were immunoreactive to anti-phosphoserine (isoform 2) and/or anti-phosphothreonine (isoform 3) antibodies. The ratio between isoforms 2 and 1 of the 30-day-old female mice was higher than that of the 20-day-old female mice. However, the ratios among the three isoforms in the male pituitary glands did not differ with age. The ratio of PPRL to isoform 1 was obviously reduced after ovariectomy (OVX), and it recovered with estrogen replacement. These results suggest that estrogen influences PRL phosphorylation in female mice.     

Caprine somatic cell nuclear transfer using in vivo matured oocytes collected by laparoscopic follicular aspiration

In vivo matured oocytes collected by laparoscopic follicular aspiration (LFA) from hormone treated female goats were used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Japanese native (Shiba) goats were used as donor females and some donor females were used repeatedly (two or three times) at intervals of a few months. To induce synchronization of estrus, a sponge containing 0.5 g of progesterone was inserted into the vagina of each goat for 14 days. These animals were also treated with follicle stimulating hormone (FSH) in a series of 8 injections over 4 days. The first FSH injection was administered on the morning of day 9 of sponge insertion. On the morning of day 13, 50 µg of gonadotropin‐releasing hormone (GnRH) was injected into each animal. Twenty‐nine hours after GnRH injection, LFA was performed. After removal of cumulus cells, collected oocytes with the first polar body were selected and enucleated for nuclear transfer. Anterior pituitary cells isolated from an adult male Shiba goat were transfected with a DNA fragment containing the enhanced green flourescent protein gene and the puromycin resistance gene. A single donor cell was inserted into the perivitelline space of each enucleated oocyte and fusion was induced with one electric pulse of 20 V for 10 µs. The SCNT goat eggs were cultured in chemically defined medium at 38.5°C in 5% CO₂, 5% O₂, 90% N₂ for 9 days. By LFA, 396 oocytes were collected from a total of 30 females. After removal of cumulus cells, 64% of them extruded the first polar body. The percentage of SCNT goat eggs produced using in vivo matured oocytes which developed to the blastocyst stage (20–21%) was significantly higher (P < 0.05) than that produced with in vitro matured oocytes (3–8%).     

Food organisms and feeding habits of larval and juvenile Japanese flounder Paralichthys olivaceus at Ohama Beach in Hiuchi-Nada, the central Seto Inland Sea, Japan

ABSTRACT:   The abundance of food organisms and feeding habits of larval and juvenile Japanese flounder were examined during the period from May to August in 1999, 2000 and 2001 at the sandy Ohama beach, the central Seto Inland Sea. The food organisms collected with a sledge net consisted of 40 families from 18 orders, dominated by mysids, crangonids and gammarids. The mean densities of mysids, crangonid shrimp ( Crangon spp.), gammarids and fish were 2.74, 6.74, 2.91 and 0.15 individuals/m², respectively. The main prey of the flounder ( n  = 202; range of total length 9.80–75.95 mm) was mysids and small crangonid shrimp (<14 mm in body length). Prey fish availability was low, as the density of fish was low. The small crangonid shrimp was abundant, and the large crangonid shrimp, which could prey on larval flounder, was not abundant. The crangonid shrimp was important not as a predator for the flounder but as prey. The flounder preferred epifaunal mysids, Nipponomysis ornata and Anisomysis ijimai , to sand-burrowing mysids, Iiella oshimai , and avoided crangonid shrimp.     

Estrogen regulates the serum level of phosphorylated prolactin in mice

Phosphorylated prolactin (PPRL) is considered to be the most quantitatively important post-translationally modified form of prolactin (PRL) in rodents. We recently detected two different types of PPRL in the mouse pituitary gland; one was phosphorylated at serine and the other was phosphorylated at serine/threonine. Furthermore, we showed that there are obvious differences in the ratios between PPRLs and non-phosphorylated PRL in the pituitary gland based on age and sex and that estrogen influences PRL phosphorylation at serine in female mice. In the present study, we examined whether estradiol (E2) increases serine PPRL in the male pituitary gland in the same manner as in the female pituitary gland and examined whether PPRL is released into serum. We first determined the relative amounts of intrapituitary PPRLs in male mice under different pharmacological conditions that increased PRL secretion. The results indicated that treatment with E2 increases serine PPRL. We then performed two-dimensional electrophoresis and immunoblotting analysis after immunoprecipitation with anti-mouse PRL antibody using male and female sera under different pharmacological conditions that increased PRL secretion. The results of this experiment indicated that there were PRLs phosphorylated at serine and serine/threonine in the female serum but not in the male serum. The levels of PPRLs in sera were greatly increased with the E2 treatment for both male and female sera. Furthermore, we examined the effect of E2 on PPRL synthesis in cultured male pituitary glands. In this experiment, we observed increased serine PPRL synthesis and stronger immunohistochemical staining of PRL cells with E2 treatment. These findings suggested that serine PPRL synthesis and secretion were influenced by estrogen.     

Dependence of self-tolerance on TRAF6-directed development of thymic stroma

The microenvironments of the thymus are generated by thymic epithelial cells (TECs) and are essential for inducing immune self-tolerance or developing T cells. However, the molecular mechanisms that underlie the differentiation of TECs and thymic compartmentalization are not fully understood. Here we show that deficiency in the tumor necrosis factor receptor-associated factor (TRAF) 6 results in disorganized distribution of medullary TECs (mTECs) and the absence of mature mTECs. Engraftment of thymic stroma of TRAF6(-/-) embryos into athymic nude mice induced autoimmunity. Thus, TRAF6 directs the development of thymic stroma and represents a critical point of regulation for self-tolerance and autoimmunity.