Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin f3 subunit to the follitropin (8 subunit (biologic Communicated by Oliver H. Lowry, February 5, 1992 ABSTRACT Follitropin (FSH) is a pituitary glycoprotein hormone that is essential for the development of ovarian follicles and testicular seminiferous tubules. FSH is used clinically to stimulate follicular maturation for in vitro fertilization and treatment of anovulatory women. One issue regarding the clinical use of FSH is its short half-life in the circulation. To address this point, we constructed chimeric genes containing the sequence encoding the C-terminal peptide of the chorionic gonadotropin 13 subunit (CG1) fused to the translated sequence of the human FSH P subunit (FSH(8). This region of CGI3 is important for maintaining the prolonged plasma half-life of human CG dimer. The presence of the C-terminal peptide sequence did not significantly affect assembly of FSH(3 with the a subunit or secretion of the dimer. In vitro receptor binding and steroidogenic activity of dimer bearing the FSHf-Cterminal peptide chimera were the same as wild-type FSH.However, both the in vivo potency and half-life in circulation of the dimer bearing either one or two C-terminal peptide units were enhanced. Dimers containing FSH(-CGI3 chimeras could serve as potent FSH agonists for clinical use, and the present strategy may have wide applications for enhancing the in vivo half-life of diverse proteins.

These data suggest that the prenatal and postnatal supplementation of bifidobacteria is effective in primary preventing allergic diseases. Some limited changes in the composition of fecal microbiota by the bifidobacterial supplementation were observed.

To elucidate the role of apoptotic cell death in human corpus luteum (CL) regression, human CL during the menstrual cycle and early pregnancy were isolated and processed for biochemical (radio-labeling) analysis of DNA integrity. Total DNA extracted from human CL of the early luteal phase contained predominantly high mol wt DNA, whereas CL of the midluteal phase exhibited the appearance of DNA cleavage into low mol wt ladders characteristic of apoptosis. Although apoptotic DNA cleavage of human CL significantly increased from the midluteal phase to the late luteal phase (P < 0.05), CL of early pregnancy did not exhibit apoptotic DNA fragmentation by biochemical analysis. In situ analysis of DNA fragmentation revealed that both large and small luteal cells exhibited DNA cleavage in human CL of the midluteal and late luteal phases and in regressive CL. The present findings suggest that 1) human luteal regression may be mediated by apoptosis; and 2) CL of early pregnancy may be rescued from luteolysis through inhibiting the occurrence of apoptotic luteal cell death.

Pituitary gonadotropin FSH acts exclusively on ovarian granulosa cells by binding to specific plasma membrane receptors. Transforming growth factors alpha and beta (TGF alpha and TGF beta), produced locally within the ovary, have been shown to regulate diverse follicle functions, although their potential role in the regulation of FSH receptors has not been assessed. Our first objective was to demonstrate developmental changes in the expression of FSH receptor gene and protein; we then analyzed the regulation of FSH receptor expression by TGF beta s and TGF alpha in cultured granulosa cells. Analysis of steady-state FSH receptor mRNA and protein levels in neonatal and prepubertal ovaries revealed the existence of two predominant FSH receptor mRNA transcripts, 7.0 and 2.5 kb in size, showing a dramatic increase between Day 15 and Day 18 of age followed by a plateau up to 27 days of age. A close parallelism in the developmental changes in FSH receptor mRNA levels and FSH receptor content was observed. Cultured granulosa cells obtained from estrogen-treated immature rats exhibited FSH receptor transcripts similar in size to those seen in whole ovaries. Treatment of granulosa cells for 48 h with TGF beta 1 increased the levels of FSH receptor mRNA for both the 7.0- and 2.5-kb transcripts in a dose-dependent manner (ED50, 1.5 ng/ml), with a maximal 4.0 +/- 0.8-fold increase over control levels observed in response to 10 ng/ml TGF beta 1. Also, TGF beta 2 was as potent as TGF beta 1 in increasing FSH receptor mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

The actions of gonadotropins on ovarian differentiation are associated with dynamic changes in gonadotropin receptor content, presumably due to modulation of receptor gene expression. The present studies used a reverse transcription-polymerase chain reaction to obtain a rat FSH receptor cDNA fragment, followed by synthesis of a labeled cRNA probe to examine the regulation of FSH receptor mRNA levels during follicular maturation, ovulation, and luteinization. Northern blot analysis of ovarian RNA with the FSH receptor probe revealed two predominant hybridization signals of 7.0 and 2.5 kilobases (kb) as well as minor signals of 4.2 and 1.8 kb. Treatment of immature rats with PMSG (10 IU) to induce follicular development resulted in increased FSH receptor mRNA levels 24 h after treatment, with a further increase at 52 h, coincident with increased [125I]FSH binding. Subsequent treatment with an ovulatory dose of hCG decreased FSH binding and receptor mRNA levels by 6 h, with a maximal inhibition at 24 h after hCG. In luteinized ovaries obtained 3 and 5 days after hCG treatment, the 7.0-kb FSH receptor mRNA increased again, but no concomitant elevation of [125I]FSH binding was detected. We recently demonstrated that FSH treatment alone is capable of inducing follicular growth and ovulation, thus providing a unique model to evaluate the effects of FSH on regulation of its receptor gene. Immature hypophysectomized estrogen-treated rats were implanted with an osmotic minipump delivering recombinant human FSH (rcFSH; 4 IU/day) to stimulate follicle growth, followed 52 h later with a single injection (20 IU) of rcFSH to induce ovulation. Stimulation of follicular growth with rcFSH increased both FSH receptor binding and mRNA levels. In contrast, the ovulatory dose of rcFSH decreased FSH binding and receptor message levels within 12 h. Thus, gonadotropin regulation of ovarian FSH receptor content during follicular growth, ovulation, and luteinization is associated with similar changes in FSH receptor message levels. Also, studies using rcFSH demonstrate that both up- and down-regulation of FSH receptor gene expression can be induced by the homologous hormone at different stages of follicle development.