Gunnar Selstam

Infertility caused by retardation of follicular development in mice with oocyte-specific expression of Foxo3a

In recent years, mammalian oocytes have been proposed to have important roles in the orchestration of ovarian follicular development and fertility. To determine whether intra-oocyte Foxo3a, a component of the phosphatidylinositol 3-kinase (PI3K) signaling pathway, influences follicular development and female fertility, a transgenic mouse model was generated with constitutively active Foxo3a expressed in oocytes. We found that the female transgenic mice were infertile, which was caused by retarded oocyte growth and follicular development, and anovulation. Further mechanistic studies revealed that the constitutively active Foxo3a in oocytes caused a dramatic reduction in the expression of bone morphogenic protein 15 (Bmp15), connexin 37 and connexin 43, which are important molecules for the establishment of paracrine and gap junction communications in follicles. Foxo3a was also found to facilitate the nuclear localization of p27kip1 in oocytes, a cyclin-dependent kinase (Cdk) inhibitor that may serve to inhibit oocyte growth. The results from the current study indicate that Foxo3a is an important intra-oocyte signaling molecule that negatively regulates oocyte growth and follicular development. Our study may therefore give some insight into oocyte-borne genetic aberrations that cause defects in follicular development and anovulation in human diseases, such as premature ovarian failure.

Cadmium-induced decrement of the LH receptor expression and cAMP levels in the testis of rats.

Cadmium (Cd) is a widespread environmental pollutant, characterized by its ability to affect various organs. Adverse effect of Cd on the testis including decreased testosterone production are well-known phenomena, but the cellular events explaining these effects have not yet been established. In the present study the initial steps of gonadotropin mediated testosterone biosynthesis were examined in vivo in rats, in relation to Cd dose and time after injection. In the dose-response experiment Male Sprague-Dawley rats received a single subcutaneous (s.c.) injection of CdCl(2) (1, 5 or 10 micromol/kg body weight) and were sacrificed 48 h after injection. A statistically significant decrease in luteinizing hormone (LH) receptor mRNA level in the testicular tissue was demonstrated at the highest dose (10 micromol/kg). In the temporal-response experiment rats were given 10 micromol/kg of CdCl(2) s.c. and sacrificed 0.48, 4.8, 48 or 144 h after injection. LH receptor mRNA levels as well as cyclic adenosine monophosphate (cAMP) levels were found to be significantly lowered at 48 and 144 h. These observations of the mechanisms whereby Cd exerts its effect on the initial steps of testosterone biosynthesis are the first from in vivo experiments.

Mono-(2-ethylhexyl) phthalate stimulates basal steroidogenesis by a cAMP-independent mechanism in mouse gonadal cells of both sexes

Phthalates are widely used as plasticizers in a number of daily-life products. In this study, we investigated the influence of mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the frequently used plasticizer di-(2-ethylhexyl) phthalate (DEHP), on gonadal steroidogenesis in vitro. MEHP (25-100 microM) stimulated basal steroid synthesis in a concentration-dependent manner in immortalized mouse Leydig tumor cells (MLTC-1). The stimulatory effect was also detected in KK-1 granulosa tumor cells. MEHP exposure did not influence cAMP or StAR protein levels and induced a gene expression profile of key steroidogenic proteins different from the one induced by human chorionic gonadotropin (hCG). Simultaneous treatment with MEHP and a p450scc inhibitor (aminoglutethimide) indicated that MEHP exerts its main stimulatory effect prior to pregnenolone formation. MEHP (10-100 microM) up-regulated hormone-sensitive lipase and 3-hydroxy-3-methylglutaryl coenzyme A reductase, suggesting that MEHP increases the amount of cholesterol available for steroidogenesis. Our data suggest that MEHP, besides its known inhibitory effect on hCG action, can directly stimulate gonadal steroidogenesis in both sexes through a cAMP- and StAR-independent mechanism. The anti-steroidogenic effect of DEHP has been proposed to cause developmental disorders such as hypospadias and cryptorchidism, whereas a stimulation of steroid synthesis may prematurely initiate the onset of puberty and theoretically affect the hypothalamic-pituitary-gonadal axis.

Synthesis of prostaglandin F2α, E2 and prostacyclin in isolated corpora lutea of adult pseudopregnant rats throughout the luteal life-span

The ability of de novo biosynthesis of prostaglandins (PGs) in individual whole corpora lutea (CL) obtained from sterile-mated adult pseudopregnant rats on different days of the luteal phase and the post-luteolytic period was evaluated. Production of PGs, progesterone and 20 alpha-dihydroprogesterone were determined after in vitro incubation of CL extirpated from Day 2 to Day 19 after mating. A time-relationship with increased accumulation of PGs in the medium was demonstrated from 18 s to 5 h, with large increments during the first 30 min. Basal accumulation of PGs in the incubation medium was highest for 6-keto-PGF1 alpha (the stable metabolite of prostacyclin) greater than PGE2 greater than PGF2 alpha greater than thromboxane B2 (TXB2) and basal accumulation of PGF2 alpha and PGE2 measured in the medium was maximal on Day 10-11 of pseudopregnancy, concomitantly with a decline in secretion of progesterone. Addition of arachidonic acid (AA) dose-dependently increased synthesis of PGs, with absolute amounts of PGE2 greater than 6-keto-PGF1 alpha greater than PGF2 alpha greater than TXB2 and addition of 14 microM indomethacin markedly inhibited accumulation of all PGs measured. Luteinizing hormone (LH, 10 micrograms/ml) stimulated progesterone secretion on all days during pseudopregnancy, but not on the post-luteolytic Day 19. LH increased PGF2 alpha, PGE2 and 6-keto-PGF1 alpha secretion on Day 13 of pseudopregnancy by 76%, 91% and 28%, respectively, but not on the other days tested. Furthermore, stimulation of PG-synthesis by addition of AA abrogated the LH-induced progesterone accumulation markedly, but only on Day 13 of pseudopregnancy. Epinephrine (5 micrograms/ml) increased production of progesterone and also PGs, but only on Day 2 of pseudopregnancy, whereas oxytocin (100 mIU/ml) was found to be without effect on progesterone as well as PG secretion on all days tested. The results of the present study demonstrates the independent ability of the rat CL to synthesize PGG/PGH2-derived prostaglandins, including the putative luteolysin PGF2 alpha. Secondly, we demonstrate that LH and AA-induced increases in PGF2 alpha and PGE2 production during the luteolytic period, may be an autocrine or paracrine mechanism involved in luteolysis.

Changes in corpus luteum content of prostaglandin F2α and E in the adult pseudopregnant rat

Abstract Conflicting reports exist regarding the source of luteolytic PGF2α in the rat ovary. To assess the quantities of different PGs, measurements of PGF2α, PGE and PGB were performed by radioimmunoassay in the adult pseudopregnant rat ovary throughout the luteal lifespan. Ovaries of 84 rats were separated by dissection into two compartments, corpora lutea of pseudopregnancy and remainder of ovary. Tissue samples were homogenized and prostaglandins extracted and determined by radioimmunoassay. During the mid-luteal and late-luteal phases, levels of PGs were significantly higher in the corpora lutea of pseudopregnancy than in the remainder of ovary. An increase of PGF2α-content in the corpus luteum was registered with peak-levels of 53.9 ± 8.5 (mean ± SEM, N=18) ng/g tissue wet weight at day 13 of pseudopregnancy. PGE-levels reached peak-values at day 11 of pseudopregnancy (271.6 ± 28.4 ng/g w w, mean ± SEM, N=12). PGB-levels were below detection limits in all compartments for all ages studied. The present study demonstrates increased availability of PGF2α in the corpus luteum during the luteolytic period, and points toward either increased luteal synthesis or luteal binding of PGF2α during the luteolytic period.

Bone formation zones in heterotopic ossifications: histologic findings and increased expression of bone morphogenetic protein 2 and transforming growth factors beta2 and beta3.

Heterotopic ossifications (HOs) formed after total endoprosthetic replacement of the hip joint were collected during revision surgery (n = 7). Tissues collected during regular hip arthroplasty (n = 12) were used as reference. Histomorphometric analysis was performed for assessment of bone formation activity in HOs and reference bone. HOs were dissected with histological guidance into three zones: formed bone, zone of active bone formation, and zone with fibrous connective and fibrocartilagineous tissue. Relative expression of the mRNA for bone morphogenetic protein 2 (BMP-2), transforming growth factor ß2 (TGF-ß2), and TGF-ß3 was determined by reverse-transcription polymerase chain reaction relative to ß-actin. Expression of all three growth factors was higher than in orthotopic bone. Similarly, the osteoid surface density was increased in HOs. The levels of all growth factors were higher in the zone of active bone formation or remodeling than in the zone of formed bone. In matured HOs, the osteoid surface density as well as mRNA levels were lower, although still significantly raised, indicating that bone formation slows down after 2 years. Immunohistochemical analysis demonstrated the presence of TGF-ß1, TGF-ß2, TGF-ß3, and BMP-2 proteins in the zone of bone formation. We conclude that bone formation after heterotopic bone induction is initially intense, slows down within 2 years, and thereupon continues as active remodeling mainly on the border of HO. Our data indicate that BMP-2, TGF-ß2, and TGF-ß3 are involved in bone formation in HO.

Acknowledgement to the 2000 Reviewers

Adam, O., München, Germany Bauer, C.P., Gaissach, Germany Berdel, D., Wesel, Germany Boeing, H., Bergholz-Rehbrücke, Germany Eder, K., Halle, Germany Elmadfa, I., Wien, Austria Erbersdobler, H., Kiel, Germany Flynn, A., Cork, Ireland Goldenberg, H., Wien, Austria Götz, M., Wien, Austria Grimm, H., Giessen, Germany Hagemeister, H., Rostock, Germany Heymsfield, G., New York, USA Krawinkel, M., Giessen, Germany Krempf, M., Nantes, France Laplace, J.P., Paris, France Lemmens, R., Wien, Austria Linseisen, J., Heidelberg, Germany Martinez, J.A., Pamplona, Spain Metges, C., Bergholz-Rehbrücke, Germany Moser, U., Basel, Switzerland Müller, M.J., Kiel, Germany Pfeuffer, E., Kiel, Germany Pietrzik, K., Bonn, Germany Pils, K., Wien, Austria Roth, H.P., Freising-Weihenstephan, Germany Rust, P., Wien, Austria Saris, W., Mastricht, Netherlands Schrezenmeir, J., Kiel, Germany Schümann, K., München, Germany Schweigert, F.J., Bergholz-Rehbrücke, Germany Seiler, W., Basel, Switzerland Thorsdottir, I., Reykjavik, Iceland Van den Berg, H., AJ Zeist, Netherlands Vasson, M.P., Clermont-Ferrand, France Vermeer, C., Mastricht, Netherlands Wernermann, J., Huddinge, Sweden Zunft, H.J., Bergholz-Rehbrücke, Germany

The acute effect of oestrogens on testosterone production appears not to be mediated by testicular oestrogen receptors

Scatchard binding analysis was performed to measure the cytoplasmic oestrogen receptor in the testis of rats. After treatment of rats with the antioestrogen tamoxifen no oestrogen receptor binding was found in testicular low speed supernatant between 12 and 96 h after treatment. Such tamoxifen-treated rats were used to study the acute effect of oestrogens on testosterone secretion, both in vivo and in vitro. Injection of oestradiol benzoate (50 microgram, 24 h prior to experiment) resulted in a significant depression of basal and LH-stimulated plasma testosterone levels in control rats and this effect was unchanged in tamoxifen-pretreated rats. In vitro, oestradiol-17 beta also inhibited the LH-induced rise in testosterone secretion by isolated testicular interstitial cells. This inhibition was not affected if the rats had been pretreated with tamoxifen. These results indicate that the inhibitory effects of exogenous oestrogens on testicular testosterone production are probably not mediated by the oestrogen receptor.

A Comparative Longitudinal Study on Sex Hormone Binding Globulin Capacity During Estrogen Replacement Therapy

Abstract. An aqueous two‐phase equilibrium partition system was used to assay SHBG‐binding capacity. Sera from groups of postmenopausal women before and during unopposed estrogen replacement therapy were analyzed. The induction of SHBG showed considerable differences between different estrogens. Ethinyl‐estradiol in a daily dose of 0.05 mg gave a 70% increase in the serum concentration of this liver derived protein. Estradiol‐17β, 2 mg daily and estrone sulphate 1.25 mg gave moderate changes, whereas estriol in different doses had no effect. SHBG induction may reflect estrogen overtreatment.

Toxic effects of the antifertility agent gossypol in male rats

In the present investigation the influence of gossypol on the male sex function and toxicology in the rat have been studied. Gossypol was injected daily for 5 weeks to adult male rats of the Sprague-Dawley strain with 1 or 10 mg/kg body wt. The low dose of gossypol (1 mg/kg body wt.) did not have any effect on the following parameters: plasma testosterone concentrations, body growth, kidney weights, sex organ weights (ventral prostate, seminal vesicle, epididymis), testicular weights, blood flow to testes, epididymides and ventral prostate as measured with the microsphere technique, intraarterial blood pressure and morphology of testis and epididymis. The plasma testosterone response to acute LH-injection was not significantly different in gossypol-treated rats when compared to control rats. The high dose of gossypol (10 mg/kg body wt) caused signs of tubular degeneration, retarded body growth, markedly reduced testosterone concentrations, involutions of the ventral prostate and seminal vesicles and gastrointestinal disturbances. After 5 weeks of treatment the mortality rate was 13%. It is concluded that gossypol is a very toxic substance in rats, since only a 10-fold increase of a non-effective dose caused serious side effects in addition to its antifertility effects.