Transgenic models to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis.

The role of FSH in gonadal tumorigenesis and, in particular, in human ovarian cancer has been debated. It is also unclear what role the elevated FSH levels in the inhibin-deficient mouse play in the gonadal tumorigenesis. To directly assess the role of FSH in gonadal growth, differentiation, and gonadal tumorigenesis, we have generated both gain-of-function and loss-of-function transgenic mutant mice. In the gain-of-function model, we have generated transgenic mice that ectopically overexpress human FSH from multiple tissues using a mouse metallothionein-1 promoter, achieving levels far exceeding those seen in postmenopausal women. Male transgenic mice are infertile despite normal testicular development and demonstrate enlarged seminal vesicles secondary to elevated serum testosterone levels. Female transgenic mice develop highly hemorrhagic and cystic ovaries, have elevated serum estradiol and progesterone levels, and are infertile, mimicking the features of human ovarian hyperstimulation and polycystic ovarian syndromes. Furthermore, the female transgenic mice develop enlarged and cystic kidneys and die between 6-13 weeks as a result of urinary bladder obstruction. In a complementary loss-of-function approach, we have generated double-homozygous mutant mice that lack both inhibin and FSH by a genetic intercross. In contrast to male mice lacking inhibin alone, 95% of which die of a cancer cachexia-like syndrome by 12 weeks of age, only 30% of the double-mutant male mice lacking both FSH and inhibin die by 1 yr of age. The remaining double-mutant male mice develop slow-growing and less hemorrhagic testicular tumors, which are noted after 12 weeks of age, and have minimal cachexia. Similarly, the double-mutant female mice develop slow-growing, less hemorrhagic ovarian tumors, and 70% of these mice live beyond 17 weeks. The double-mutant mice demonstrate minimal cachexia in contrast to female mice lacking only inhibin, which develop highly hemorrhagic ovarian tumors, leading to cachexia and death by 17 weeks of age in 95% of the cases. The milder cachexia-like symptoms of the inhibin and FSH double-mutant mice are correlated with low levels of serum estradiol and activin A and reduced levels of aromatase mRNA in the gonadal tumors. Based on these and our previous genetic analyses, we conclude that elevated FSH levels do not directly cause gonadal tumors. However, these results suggest FSH is an important trophic modifier factor for gonadal tumorigenesis in inhibin-deficient mice.     

Novel functional hepatocyte cell line derived from spontaneous dwarf rat: Model of growth hormone function in vitro

Currently there is no good hepatocyte model for studying growth hormone (GH) function that reflects its normal physiological roles. Here we report the establishment of a functional hepatocyte cell line, SDRL-1, from the liver of young male spontaneous dwarf rats (SDR), with isolated GH deficiency. This line has been maintained in Dulbecco's Modified Eagle Medium (DMEM)/F12 medium supplemented with 10% fetal bovine serum (FBS) with retention of a near diploid karyotype for extended periods of time. When grown as a monolayer sheet, it displayed a pavement-like appearance and contact inhibition. These cells have a poorly developed rough endoplasmic reticulum (r-ER), few mitochondria and glycogen granules, and produce a small amount of albumin and α-fetoprotein, that is enhanced when grown on a collagen gel sponge. Human recombinant GH stimulated JAK2 and STAT5b tyrosine phosphorylation and IGF-I production in a concentration-dependent manner. When the cells were cultured with GH-supplemented medium, the number of mitochondria and glycogen granules increased together with the r-ER and Golgi apparatus. A number of microvilli were observed on the surface of the cultured cells, further suggesting that this cell line is composed of normally functioning hepatocytes. In summary, we established a novel hepatocyte cell line (SDRL-1), that appears to display normal function, which we propose can serve as a good in vitro model for studying GH-target organ interactions.     

In vivo effects of follicle-stimulating hormone on testicular macrophages

The effect of an intraperitoneal injection of various hormones on the incorporation of amino acids and uridine into acid-precipitable material by subsequently isolated testicular macrophages was investigated. It was found that treatment with follicle-stimulating hormone (FSH), but not luteinizing hormone (LH) or insulin, significantly increased incorporation of amino acids into secreted (but not cellular) protein and uridine incorporation into cellular RNA in a dose-dependent manner. Maximal responsiveness was observed at a dose of 50 micrograms of the hormone. These studies demonstrate that FSH has an action on testicular macrophages in vivo.     

Periodate oxidation studies of human pituitary follicle-stimulating hormone

A preparation of human pituitary follicle-stimulating hormone was subjected to periodate oxidation, borohydride reduction and acid hydrolysis. Comparison of the analysis of the remaining intact carbohydrate and amino acid units with the analyses of the original material and identification of the carbohydrate fragments permit some structural assignments to the molecule of follicle-stimulating hormone. The results of radioimmunological assay of fragments of the molecule of follicle-stimulating hormone suggest that, although the carbohydrate component is essential for biological activity, it is not a requirement for immunological activity, which appears to be a function of the protein moiety.     

Effects of in vivo administration of testosterone propionate on in vitro production of follicle-stimulating hormone and luteinizing hormone by pituitaries of pony mares

The in vitro incorporation of [3H]leucine into immunoprecipitable follicle-stimulating hormone (FSH) and luteinizing hormone (LH) was assessed for pituitaries from pony mares treated with testosterone propionate (TP) or oil (controls). Mares were treated every other day with TP (n = 4) at 350 micrograms/kg of body weight or with an equivalent volume of oil (n = 4). One day following the sixth injection of TP, each mare received an intravenous injection of gonadotropin releasing hormone (GnRH) at 1.0 micrograms/kg body weight and was bled frequently for 4 h. Treatment of mares with TP reduced FSH (P less than 0.05) and LH (P less than 0.01) concentrations in daily blood samples and increased (P less than 0.01) the amount of FSH secreted in response to GnRH compared with control mares. Incorporation of [3H]leucine into immunoprecipitable FSH was also greater (P less than 0.01) in pituitaries from TP-treated mares compared with control mares on both a per mg tissue and per anterior pituitary basis. The amount of LH secreted after GnRH, the amount left in the pituitary and the incorporation of [3H]leucine into LH were not affected by treatment. These results confirm earlier conclusions drawn from indirect evidence that androgens increase the production of FSH in the mare.     

Ovulations in rat ovaries perfused in vitro with follicle-stimulating hormone

Using the model of the isolated perfused rat ovary, we have found that highly purified ovine follicle-stimulating hormone (FSH) preparations cause ovulation and that this effect is not due to luteinizing hormone (LH) contamination. Ovine FSH-13 at a concentration of 1.5 mU/ml induced ovulations in all perfused ovaries (8.8 +/- 2.3 ovulations/ovary), as did a more purified preparation, ovine FSH-211B, at concentrations of 0.5 mU/ml (15.0 +/- 6.4 ovulations/ovary) and 5 mU/ml (11.3 +/- 2.6 ovulations/ovary). This ovulation-inducing effect of FSH is accompanied by a marked stimulation of estradiol levels in the perfusion medium without stimulation of progesterone levels. Furthermore, a purified rat FSH preparation (15 mU/ml) also induced ovulation in all ovaries (13.8 +/- 2.2 ovulations/ovary) as well as a stimulation of both estradiol and progesterone in the medium. These data clearly confirm the direct ovulatory effect of FSH on the ovary.     

In vitro and in vivo studies on cytodifferentiation of pituitary clonal cells derived from the epithelium of Rathke's pouch

Summary A clonal strain of anterior pituitary cells was derived from Rathke's pouch of the rat. These cells were shown to secrete ACTH, growth hormone and prolactin but no glycoprotein hormones, when grown in vitro. Cells from the 2A8 clone were implanted for one month under kidney capsules or into hypothalami of hypophysectomized female rats. Under the kidney capsule, prominent prolactin cells and poorly developed cells of other types were differentiated as seen in usual pituitary grafts. In hypophysiotrophic areas of the hypothalamus, the grafts were cytodifferentiated into various types of anterior pituitary cells with rich vascularization. These cells had the ultrastructural features indicative of hormone secretion. Increases in body and ovarian weights reflected the secretion of somatotrophic and gonadotrophic hormones. The results obtained indicate that implants of 2A8 clonal cells may differentiate into all types of anterior pituitary cells under the influence of hypothalamic hormones or perhaps some unknown factors present in the general systemic circulation of the rat.Supported by USPHS Grant AM 12583The authors wish to thank Mrs. Martha Castilleja and Mrs. Pauline Polette for their skillful technical assistance     

Comparative in vitro and in vivo studies on long-wavelength photosensitizers derived from bacteriopurpurinimide and Bacteriochlorin p6: fused imide ring enhances the in vivo PDT efficacy

In situ conversion of bacteriochlorophyll-a, present in Rhodobacter sphaeroides (Rb. sphaeroides) gave bacteriopurpurin-18 in modest yield, which in a sequence of reactions was converted into two series of bacteriochlorins: bacteriopurpurinimide and bacteriopurpurin p6 with and without a fused imide ring system, respectively. To determine the effect of overall lipophilicity in photosensitizing efficacy, these bacteriochlorins were independently reacted with HBr gas and subsequently treated with various alkyl alcohols to afford the corresponding alkyl ether derivatives as diastereomeric mixtures (the R- and S-isomers were obtained in almost equal ratios). Between the two series of bacteriochlorins, the bacteriopurpurinimides containing a fused imide ring system were found to be more effective in vivo (C3H mice bearing RIF tumors). To investigate the effect of the presence of the chiral center at position 3 of the most effective purpurinimide 9 [3(1'-heptyloxy)ethyl-3-deacetyl-bacteriopurpurin-18-N-hexylimide propyl ester], the acetyl group was replaced with a hydroxymethyl substituent and converted into 3(1'-decyloxy)methyl-3-deacetyl-purpurin-18-N-hexylimide methyl ester 26 with a similar lipophilicity. Interestingly, compared to 26, the bacteriopurpurinimide 9 was found to be more effective, suggesting that the chiral center at position 3 certainly plays an important role in photosensitizing activity. Among a series of alkyl ether analogues, between the PDT efficacy and the lipophilicity (log P and log D) calculated by computational methods (PALLAS program), a parabolic relationship was observed to some extent. However, it was limited to a particular series, e.g., compounds with similar log P values between bacteriopurpurinimides and bacteriochlorin e6 did not produce similar in vivo efficacy. As expected, within a series, a linear relationship was observed between the log P values and the HPLC retention times of the photosensitizers. Some of the mitochondrial localized photosensitizers showed a significant peripheral benzodiazepine binding (PBR) affinity. However, limited correlation between PBR binding affinity and in vivo PDT efficacy was observed. Compared to the naturally occurring bacteriochlorophyll-a, the bacteriopurpurinimides with fused imide ring system showed higher in vitro/in vivo stability. In contrast to methyl pyropheophorbide-a, the ester functionalities in bacteriopurpurinimide did not convert into the corresponding carboxylic acid by the enzyme esterases.     

Impact of long-term hormone replacement therapy on in vivo and in vitro markers of lipid oxidation

Postmenopausal hormone replacement therapy (HRT) with estrogen has been suggested to inhibit oxidation of low-density lipoprotein (LDL) in vitro, but progestins may oppose this effect. We studied whether estrogen HRT and combined HRT with estrogen and progestin differ in their ability to resist in vivo and in vitro oxidation of lipids. Study group included 15 women on oestradiol valerate (mean age 56 years, treatment duration 10.5 years) and 15 women on combined HRT with oestradiol valerate and levonorgestrel (mean age 58 years, treatment duration 11.3 years). In addition to lipid and apolipoprotein concentrations, the lagtime of LDL to oxidation, the rate of the propagation phase and the maximum concentration of conjugated dienes were recorded as indices of LDL susceptibility to copper-induced oxidation in vitro. As an in vivo marker of oxidative stress we measured 24-h excretion of urinary 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha). All measurements were done after long-term HRT (baseline), after 4 weeks pause and again 3 weeks after reintroduction of HRT. High-density lipoprotein (HDL) cholesterol and apolipoprotein AI concentrations were significantly higher and LDL to HDL ratio significantly lower after long-term oestradiol valerate therapy than after combined therapy. Simultaneously, the triglyceride and lipoprotein (a) levels were higher in the estrogen group. Susceptibility of LDL to oxidation and the level of 8-iso-PGF2alpha were similar in both groups at all measurement points, and treatment group was not a statistically significant determinant of these markers at baseline. According to these results, estrogen and combined HRT do not differ in their abilities to oppose LDL oxidation in vitro or systemic oxidative stress in vivo, but have differential effects on blood lipids.     

Effect of follicle-stimulating hormone and luteinizing hormone during bovine in vitro maturation on development following in vitro fertilization and nuclear transfer

The effects of luteinizing hormone (LH) (0, 100, 10,000 lU/ml) and follicle-stimulating hormone (FSH) (20 μg/ml) supplementation during in vitro maturation of slaughterhouse-derived oocytes on polar body formation and embryo development subsequent to in vitro fertilization and nuclear transfer were evaluated. Go-nadotropin supplementation of maturation medium in the presence of serum neither enhanced the proportion of oocytes forming a polar body nor significantly affected development following in vitro fertilization or nuclear transfer, except at the highest LH concentration. A very high concentration of LH (10,000 lU/ml) significantly decreased polar body formation, initial cleavage, and blastocyst development (P < 0.05). © 1993 Wiley-Liss, Inc.     

Structural investigation of the carbohydrate element of human pituitary follicle-stimulating hormone by methylation analysis

A preparation of human follicle-stimulating hormone has been subjected to methylation analysis by using the methyl sulphinyl carbanion-dimethyl sulphoxide-methyl iodide method. The hydrolysis products of the methylated glycoprotein were reduced, acetylated, analysed by gas-phase chromatography and mass spectrometry and identified by comparison with standards. Methylation analysis demonstrated that (1) the d-galactose, mannose, fucose and 2-amino-2-deoxyglucose units exist in the pyranose forms, (2) the 2-amino-2-deoxyglucose units are N-acetylated, (3) the fucopyranose units occupy terminal non-reducing positions, (4) the d-galactopyranose units are linked in the 1- and 2-positions, (5) the mannopyranose units exist in three forms, some as terminal non-reducing residues, some as 1,6-linked residues and some as 1,3,4-linked branch points, and (6) the 2-acetamido deoxyglucopyranose units are 1,6-linked. These structural assignments are compared with other data previously obtained for the carbohydrate moieties of follicle-stimulating hormone.