Diagnostic Accuracy of the Abbott SD Bioline MPT64 antigen test for identification of MTB Complex in a U.S. Clinical Mycobacteriology Laboratory.

Identification of the Mycobacterium tuberculosis complex (MTBC) from culture and differentiation from other non-tuberculous mycobacterial species is required for rapid diagnosis and accurate treatment. However, gaps exist in culture-based identification of acid-fast bacilli (AFB) positive cultures for rapid rule-out of MTBC in the United States. The SD Bioline™ MPT64 (Abbott Inc, South Korea) lateral flow assay (LFA) has high sensitivity and specificity for the detection of MTBC in liquid culture but has not been evaluated in a clinical mycobacteriology laboratory in the United States. We conducted a diagnostic accuracy study of this LFA for detection of MTBC versus NTMs on AFB positive cultures. A total of 362 tests were performed, with a sensitivity and specificity of 100 % (362/362) across all tests. The SD Bioline MPT64 assay provides accurate test results with AFB-positive liquid cultures and could fill the current gap for rapid rule-out of MTBC in U.S.-based clinical laboratories.

Simulated physiological oocyte maturation (SPOM): a novel in vitro maturation system that substantially improves embryo yield and pregnancy outcomes.

BACKGROUND: Oocyte in vitro maturation (IVM) reduces the need for gonadotrophin-induced ovarian hyperstimulation and its associated health risks but the unacceptably low conception/pregnancy rates have limited its clinical uptake. We report the development of a novel in vitro simulated physiological oocyte maturation (SPOM) system. METHODS AND RESULTS: Bovine or mouse cumulus-oocyte complexes (COCs) were treated with cAMP modulators for the first 1-2 h in vitro (pre-IVM), increasing COC cAMP levels ∼100-fold. To maintain oocyte cAMP levels and prevent precocious oocyte maturation, COCs were treated during IVM with an oocyte-specific phosphodiesterase inhibitor and simultaneously induced to mature with FSH. Using SPOM, the pre-IVM and IVM treatments synergized to increase bovine COC gap-junctional communication and slow meiotic progression (both P < 0.05 versus control), extending the normal IVM interval by 6 h in bovine and 4 h in mouse. FSH was required to complete maturation and this required epidermal growth factor signalling. These effects on COC had profound consequences for oocyte developmental potential. In serum-free conditions, SPOM increased bovine blastocyst yield (69 versus 27%) and improved blastocyst quality (184 versus 132 blastomeres; both P < 0.05 versus standard IVM). In mice, SPOM increased (all P < 0.05) blastocyst rate (86 versus 55%; SPOM versus control), implantation rate (53 versus 28%), fetal yield (26 versus 8%) and fetal weight (0.9 versus 0.5 g) to levels matching those of in vivo matured oocytes (conventional IVF). CONCLUSIONS: SPOM is a new approach to IVM, mimicing some characteristics of oocyte maturation in vivo and substantially improving oocyte developmental outcomes. Adaption of SPOM for clinical application should have significant implications for infertility management and bring important benefits to patients.

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus).

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus-oocyte complexes (COCs) were isolated from large (>1.5mm) and small (0.7-1.5mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 +/- 9.3 versus 6.4 +/- 2.7, mean +/- S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.

Growth-inhibitory effects of CD40 ligand (CD154) and its endogenous expression in human breast cancer.

CD40 binding produces multifaceted growth signals in normal and malignant B cells, whereas its physiological role is less well characterized in epithelial cancers. We examined the growth outcome of CD40 ligation in human breast cancer cells, using CD40+ (T47D and BT-20) and CD40-negative (MCF-7, ZR-75-1) cell lines as defined by flow cytometric analysis, immunohistochemistry, and reverse transcription-PCR. Treatment with the soluble recombinant CD40 ligand (CD40L) molecules gp39 or CD40L-trimer significantly reduced [3H]thymidine uptake in BT-20 and T47D cells by up to 40%, but did not affect the growth of CD40-negative MCF-7 or ZR-75-1 cells. Similarly, significant growth inhibition was observed after co-incubation with CD40L-transfected murine L cells (55.0 +/- 8.9%, P < 0.001) that express membrane CD40L constitutively, or with paraformaldehyde-fixed, CD3+ CD40L+ PBLs from three different HLA-mismatched donors (39.7 +/- 3.7%, P < 0.01). Untransfected L cells and non-CD40L-expressing lymphocytes did not produce significant growth inhibition. The in vivo antitumorigenic effects of CD40L were examined using a s.c. severe combined immunodeficient-hu xenograft model. Pretreatment with two different soluble recombinant CD40L constructs (CD40L and gp39) produced similar xenograft growth-inhibitory effects [67 +/- 24% (n = 4), and 65 +/- 14% (n = 8) inhibition, respectively], which were reversed by co-treatment with the CD40L-neutralizing antibody LL48. In vitro analysis indicated that CD40L-induced growth inhibition was accompanied by apoptotic events including cell shrinkage, rounding, and detachment from the adherent T47D culture monolayer. Thirty-one and 27% of gp39-treated T47D and BT-20 cells underwent apoptosis, respectively, as compared with 56 and 65% from the same cell lines after treatment with the Fas agonistic antibody CH-11. An up-regulation of the proapoptotic protein Bax in T47D and BT-20 cells was observed, which indicated that this Bcl-2 family member may contribute to this growth-inhibitory effect. To explore the clinical relevance of CD40L-CD40 interaction, retrospective immunohistochemical analysis was carried to characterize in situ CD40- and CD40L-expression in breast cancer patient biopsies. All of the infiltrating ductal (5 of 5 cases tested) and lobular (4 of 4 cases) breast carcinomas, carcinomas in situ (6 of 6 cases), and mucinous carcinoma tested (1 case) expressed CD40. Varying proportions of tumor cells also expressed CD40L in the majority of infiltrating ductal (3 of 5 cases) and lobular (3 of 4 cases) carcinomas, and carcinomas in situ (4 of 6 cases), as determined by immunohistochemistry and validated by RT-PCR detection of the CD40L message in only CD40L positive-staining cases. Tumor infiltrating mononuclear cells from infiltrating carcinomas and carcinomas in situ expressed CD40 (10 of 10 cases), but less commonly CD40L (1 case of infiltrating lobular carcinoma, 2 cases of carcinoma in situ). Our findings indicate that the CD40 signaling pathway is active in human breast carcinoma cells. However, tumor-infiltrating lymphocytes from primary tumor tissues may be limited in their capacity to directly modulate tumor growth through the CD40L-CD40 loop.

Inhibition of ovarian estradiol-17beta secretion by luteinizing hormone in prepubertal, pregnant mare serum-treated rats.

Serum estradiol-17beta levels, elevated prior to the luteinizing hormone (LH) surge, decline abruptly following the release of endogenous LH or the injection of exogenous LH. To investigate the mechanism of this decline, bovine LH (NIH-LH-B8) was administered to immature rats, in which follicular maturation and estrogen biosynthesis were induced by a non-ovulating dose of pregnant mare serum gonadotropin (PMS). Serum and ovarian estradiol-17beta concentrations fell detectably by 4h, and reached levels around 20% of the controls by 8h after iv injection of 10 mug LH. Concomitant decreases occurred in ovarian androgen concentrations, following an initial rise, and in the in vitro ovarian testosterone aromatizing enzyme activity. The LH-induced inhibition of the aromatase activity was found to be of a non-competitive type. It is proposed that two enzyme systems are inhibited as a result of the LH treatment: the C17,20-lyase and the C19 androgen aromatase, thereby leading to decreased concentrations of estrogens in the ovaries and blood.

Androgen production by theca and granulosa isolated from proestrous rat follicles.

Theca and granulosa isolated from proestrous rat ovarian follicles were cultured for 72 h in the presence or absence of highly purified luteinizing hormone (LH, 0.1 microng/ml) and/or follicle-stimulating hormone (FSH, 0.1 microng/ml). Medium was collected and replaced at 3, 6, 12, 24, 48, and 72 h of culture and measured for testosterone by radioimmunoassay. Pieces of isolated theca secreted androgen; androgen production was greatest during the first 12 h of culture. Addition of highly purified LH to the culture medium produced a significant increase (P less than 0.001) in the thecal androgen secretion, while addition of highly purified FSH had no significant effect. Addition of LH + FSH to culture medium produced the same effect as addition of LH alone. The response of the theca to LH was dose-dependent with doses of 0.01 microng/ml or greater eliciting a maximum response. The addition of eoxgenous progesterone (5 x 10(-7)M) to culture medium had no effect on thecal androgen production. Thecal androgen secretion was the same in the presence or absence of fetal calf serum. Since the testosterone antibody used was not entirely specific for testosterone, testosterone and the cross-reacting androgen, 17beta-hydroxy-5alpha-androstan-3-one (DHT), were chromatographically isolated from samples and assayed separately. The androgen measured in culture medium was found to consist primarily of testosterone; DHT was present in much lower concentrations. Granulosa cells isolated from the same follicles as the theca and grown in monolayer culture produced negligible amounts of androgen. It is concluded that the theca is the site of follicular androgen production and that LH regulates androgen secretion by rat ovarian follicles. The results suggest that the theca provides the androgen precursor needed for follicular estradiol-17beta synthesis.

Androgens augment FSH-induced progesterone secretion by cultured rat granulosa cells.

Granulosa cells have been isolated from ovaries of estrogen-treated immature intact and hypophysectomized rats, and have been maintained in culture in a chemically-defined medium. Progesterone secretion by these cells was testosterone or 17beta-OH-5alpha-androstan-3-one (DHT), progesterone secretion was low or undetectable. However, the addition of testosterone or DHT together with FSH caused a dramatic 8- to 19-fold increase over that caused by FSH alone. On the other hand, luteinizing hormone (LH) alone had no effect on progesterone secretion, but produced a small stimulation when added together with testosterone. These results demonstrate synergism between androgens and FSH in the control of progesterone secretion by granulosa cells in culture.

Stimulation of aromatization of exogenous and endogenous androgens in ovaries of hypophysectomized rats in vivo by follicle-stimulating hormone.

The role of follicle-stimulating hormone (FSH) in the regulation of estrogen biosynthesis in vivo has been investigated in immature hypophysectomized rats, utilizing uterine weight and histologic responses, and ovarian estradiol-17beta concentrations as indicators of estrogen secretion. A highly purified FSH preparation produced only borderline increases in uterine weights and in ovarian estradiol-17beta contents when administered alone at 2.5 mug/day for 3 days. Testosterone or androstenedione (5 mg/day), when administered in the absence of FSH, produced typically androgenic stimulation of uteri, and did not increase ovarian estradiol-17beta concentrations. When administered concomitantly with FSH, the uterine weight-stimulating activity of these aromatizable androgens was substantially increased, accompanied by marked hypertrophy of the endometrial mucosal cells, and ovarian estradiol-17beta concentrations were increased 20- to 200-fold. The administration of the non-aromatizable androgen, 17beta-OH-5alpha-androstan-3-one (DHT) (5 mg/day, by itself produced uterine weight increases similar to those caused by testosterone alone; however, no evidence of increased estrogen secretion resulted from the combined treatment of DHT wtih FSH. A highly purified luteinizing hormone (LH) preparation was equally as effective as exogenous androstenedione in increasing ovarian concentrations of immunoreactive androgen (testosterone + DHT) but evoked none of the above signs of estrogen secretion unless administered together with FSH. The weights of ovaries were not affected by the administration of LH or of any of the androgens by themselves, but were approximately doubled by FSH alone. Ovarian weights were increased still further when FSH was administered concomitantly with LH, testosterone , or androstenedione, but not with DHT. It is concluded that FSH and LH regulate ovarian estrogen secretion in vivo by acting at biochemically distinct sites--LH stimulating the synthesis of C19-steroids, which are then converted to estradiol-17beta under specific stimulation by FSH.

Loss of uterine luminal fluid in the rat: relative importance of changing peripheral levels of estrogen and progesterone.

Experiments were conducted to determine the relative importance of the decline in circulating estrogen levels, as opposed to the increase in progesterone levels, in the hormonal control of the loss of luminal fluid from the uterus of the rat, an event which usually occurs early on the morning of estrus. Silastic capsules containing crystalline estradiol-17beta (E2) were implanted subcutaneously in ovariectomized immature rats. Approximately 64 h later, the capsules were either removed (E2 withdrawal) to mimic the fall in peripheral estrogen levels occurring at proestrus, or left in place (continuous E2), and, at the same time, progesterone or oil was administered. Fifteen hours later, irrespective of whether E2 was withdrawn or not, 2 mg progesterone reduced uterine luminal fluid accumulation to levels usually seen in intact animals on the morning of estrus; however, the loss of fluid occurred slightly sooner if E2 was withdrawn when progesterone was administered. By itself, E2 withdrawal resulted in only a small decrease in uterine luminal fluid by 15 h, even though serum E2 levels had fallen to less than 1.5 pg/ml by this time. This loss was brought about by escape of the fluid through the cervix rather than by its reabsorption from the lumen. The dose-response relationships between progesterone and uterine luminal fluid accumulation indicated that when E2 was withdrawn, a smaller amount of progesterone brought about the loss of uterine luminal fluid accumulation than when E2 was continuous. These results suggest that increased progesterone secretion on the afternoon of proestrus is essential for the loss of uterine luminal fluid, and that the decline in estrogen secretion at this time may be of importance by allowing the progesterone to be more effective.

Interactions of a phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine, with prostaglandin E2, follicle-stimulating hormone, luteinizing hormone, and dibutyryl cyclic 3',5'-adenosine monophosphate (cAMP) in cAMP and steroid production by neonatal rat ovaries in vitro.

The development of responsiveness to prostaglandin E2 (PGE2), FSH, LH, and [Bu]2cAMP was examined in whole ovaries isolated from neonatal Sprague-Dawley rats on days 0 (birth), 2, 4, or 6 postpartum. Pairs of ovaries were incubated with these stimuli in the absence or presence of 3-isobutyl-1-methyl xanthine (MIX), a potent phosphodiesterase inhibitor, and accumulations in the medium of cAMP, androstenedione, and estradiol were measured. PGE2 stimulated marked cAMP accumulation on day 0 whereas similar responses to FSH and LH did not develop until days 2 and 4, respectively. No cAMP accumulation was detectable in the absence of MIX. Ovaries gradually acquired the ability to produce both cAMP and steroids in response to FSH and LH over the first postnatal week. No steroid accumulation was measurable in incubations conducted on days 0 or 2; however, steroidogenesis was stimulable in day-4 ovaries by (Bu)2cAMP. PGE2, FSH, and LH also stimulated steroid accumulation on day 4, but only when MIX was present in the incubation, suggesting that high levels of endogenous cAMP can also lead to steroid production. By day 6, all stimuli elicited steroid accumulation in a dose-dependent fashion. MIX potentiated the responses to lower doses of these stimuli but not to the higher doses at this age. In the absence of MIX, LH was approximately 100 times more potent than FSH in stimulating steroid production; however, the two gonadotropins were nearly equipotent in this regard when MIX was present in the incubation. These results support the notion that a cAMP-sensitive steroidogenic apparatus is present in the rat ovary as early as the fourth day postpartum. Because of the marked effects of MIX on gonadotropin-induced steroidogenesis, it may be that modulation of phosphodiesterase activity is one way by which steroidogenesis is regulated in the neonatal rat ovary.

Site of action of androgens on follicle-stimulating hormone-induced aromatase activity in cultured rat granulosa cells.

This paper describes experiments on cultured granulosa cells isolated from ovaries of immature rats designed to locate the site of action of androgens on FSH-induced aromatase activity. Treatment of cells during a 36-h induction period with (Bu)2cAMP, 8- BrcAMP , FSH, prostaglandin E2, or cholera toxin resulted in induction of aromatase activity measured as 17 beta-estradiol accumulation during a 6-h test period with testosterone (5 X 10(-7) M) added to medium as substrate. Presence of testosterone (5 X 10(-7) M) during the induction period enhanced the effects of FSH, cholera toxin, and prostaglandin E2 on aromatase activity, but not those of the cAMP analogs. The effects of culturing and steroids on responsiveness of granulosa cells to FSH (measured as FSH-stimulated cAMP production during a 1-h test period) were examined. The data showed that culturing in medium alone for 36 h resulted in a decrease in the ability of FSH to stimulate cAMP production when compared to that of freshly isolated cells. After culture with testosterone (5 X 10(-7) M), dihydrotestosterone (DHT) (5 X 10(-7) M), or 17 beta-estradiol (5 X 10(-7) M), responsiveness was at least partially restored. After treatment with progesterone (5 X 10(-7) M), FSH stimulation of cAMP production was not significantly different from that of cells cultured in medium alone. Hydroxyflutamide (5 X 10(-5) M), an antiandrogen known to block androgen-receptor interaction, abolished the effect of DHT and depressed the effect of testosterone on responsiveness of granulosa cells to FSH. Cells treated for 36 h with testosterone (5 X 10(-7) M) bound significantly more [125I]iodo-FSH than cells cultured in medium alone. Although DHT (5 X 10(-7) M) slightly increased FSH binding, the effect was not statistically significant. These results suggested that androgens regulate granulosa cell aromatase activity not only as substrates, but also by acting at a site before cAMP production (possibly at the level of the FSH receptor) in the control of FSH-induced enzyme activity.

Site of androgen inhibition of follicle-stimulating hormone-stimulated progesterone production in porcine granulosa cells.

In granulosa cells derived from medium-sized porcine follicles, certain androgens have been shown to inhibit FSH-stimulated progesterone synthesis. To determine the site at which this inhibition takes place, the effects of androgens on FSH- and (Bu)2cAMP-stimulated pregnenolone and progesterone syntheses were examined. Granulosa cells were isolated from 4- to 6-mm follicles and cultured for 24 h in modified Eagle's Minimum Essential Medium, alone or with FSH (1 microgram/ml) or (Bu)2cAMP (0.5-4 mM) in the presence or absence of androstenedione or testosterone. (Bu)2cAMP stimulated progesterone production in a dose-dependent manner. Testosterone (5 microM) had a slight, but nonsignificant, inhibitory effect on basal progesterone production, but significantly inhibited the synthesis of progesterone in the presence of (Bu)2cAMP, suggesting that testosterone inhibits progesterone synthesis at a step distal to cAMP formation. In the absence of FSH, granulosa cells produced substantial quantities of pregnenolone. FSH caused a 3-fold stimulation of pregnenolone synthesis. The addition of androstenedione or testosterone (5 microM) markedly increased pregnenolone accumulation in FSH-treated cultures. To determine at what step androgens affected FSH-stimulated pregnenolone production, granulosa cells were cultured with (Bu)2cAMP and/or testosterone for 24 h. (Bu)2cAMP stimulated pregnenolone synthesis in a dose-dependent manner. Testosterone (5 microM) significantly increased pregnenolone synthesis in response to (Bu)2cAMP, suggesting that androgens acted at a step distal to cAMP formation. Since these concentrations of androgens markedly inhibited FSH-stimulated progesterone production by these preparations, these results suggest that androgens may affect the conversion of pregnenolone to progesterone.

Porcine oocytes release cumulus expansion-enabling activity even though porcine cumulus expansion in vitro is independent of the oocyte.

In the mouse, the oocyte secretes a factor that enables cumulus cells to undergo expansion in response to epidermal growth factor (EGF) or FSH while expansion of the porcine cumulus oophorus has been shown to be independent of the oocyte. This study was undertaken to confirm independence of the porcine cumulus oophorus of the oocyte for its expansion and to determine if the porcine oocyte secretes the putative cumulus expansion-enabling factor that is required for FSH-stimulated mouse cumulus expansion. Porcine oocyte-cumulus cell complexes (P-OCC), oocytectomized oocyte-cumulus cell complexes (P-OOX) and intact clumps of cumulus cells (P-CCC) were cultured at 39 C in TCM-199 medium containing EGF (1 ng/ml) or FSH (1.5 micrograms/ml). After 24h culture periods, cumulus expansion was scored on an arbitrary scale of 0 to +4. EGF stimulated similar cumulus expansion (expansion score +3 compared to 0 for controls) in all the three groups. FSH stimulated cumulus expansion in significantly higher number of complexes in all groups compared to EGF. However, there was no difference in cumulus expansion among the three groups with EGF or FSH. To determine the production of the factor by the porcine oocyte, isolated clumps of cumulus cells of the mouse (M-CCC) were cocultured with porcine denuded oocytes (P-DO) in TCM-199 with or without 1.5 micrograms/ml FSH at 37 C for 24h. No expansion was observed when M-CCC were cultured alone in the presence of FSH or cocultured with P-DO in the absence of FSH. However, coculture of M-CCC with P-DO in the presence of FSH resulted in expansion of M-CCC similar to that observed in intact mouse oocyte-cumulus cell complexes (M-OCC) in response to FSH. These studies indicate that even though porcine cumulus expansion in vitro is not dependent on the oocyte, the porcine oocyte is capable of secreting cumulus expansion-enabling factor(s).

Mevinolin (lovastatin) inhibits androstenedione production by porcine ovarian theca cells at the level of the 17 alpha-hydroxylase:C-17,20-lyase complex.

Mevinolin, putatively a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme-A reductase, was used to assess the contribution of de novo synthesized cholesterol to androgen production by ovarian thecal cells in vitro. Enzymatically dispersed thecal cells from 3- to 6-mm follicles of prepubertal gilts were incubated at 150,000 cells/ml with a maximally effective dose of LH (250 ng/ml) for 24 h. Mevinolin (3-50 microM) caused dose-dependent inhibition of androstenedione production. Addition of 25-hydroxycholesterol (0.025-25 microM) failed to restore androstenedione production to levels seen in the absence of mevinolin, suggesting an additional site of action of mevinolin beyond 3-hydroxy-3-methylglutaryl coenzyme reductase. The site of this inhibitory effect was determined by measuring steroid products formed in the presence of relevant steroid precursors. Mevinolin (12 microM) inhibited the production of 17 alpha-hydroxyprogesterone from progesterone and that of androstenedione from 17 alpha-hydroxyprogesterone, while 25-hydroxycholesterol to progesterone and pregnenolone to progesterone conversions were unimpaired. That mevinolin did not affect 3 beta-hydroxysteroid dehydrogenase:delta 5-delta 4-isomerase reactions was confirmed by demonstrating that conversions of pregnenolone, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone to progesterone, 17 alpha-hydroxyprogesterone, and androstenedione, respectively, were not affected by 12 microM mevinolin. These results indicate that mevinolin has an additional inhibitory action at the level of the 17 alpha-hydroxylase:C-17,20-lyase complex. The degree of inhibition of androstenedione production was not decreased with increased concentrations of progesterone or 17 alpha-hydroxyprogesterone substrate, suggesting that the inhibition was not competitive in nature. As the dose of mevinolin was increased up to 50 microM, progesterone accumulation was unaffected, but pregnenolone concentrations in medium greatly increased. While the mechanism of this effect is unclear, this finding suggests that preformed intracellular cholesterol, rather than that synthesized de novo, is supplying steroidogenic substrate in these cells.

Stimulatory action of follice-stimulating hormone on estradiol-17 beta secretion by hypophysectomized rat ovaries in organ culture.

Ovaries of rats explanted in organ culture 2-3 days after hypophysectomy at 26 days of age synthesize ans secrete estradiol-17beta into the culture medium at approximately linear rates for 48 h. Addition of testosterone (5 times 10- minus 7M) to the culture medium occasionally caused small increases of up to 50 percent in rate of estradiol production. Significant increases (60-200 percent) in estradiol secretion resulted from addition of two different FSH preparations, in concentrations as low as 0.25 mug/ml in the absence of testosterone. In the presence of testosterone, the same FSH preparation caused much more marked increases in estradiol secretion of up to 900 percent. In the absence of testosterone, two different luteinizing hormone preparations failed to increase estradiol secretion significantly, although in the presence of testosterone, small increases were sometimes observed. It is concluded that FSH regulates estradiol biosynthesis in the hypophysectomized rat ovary by a specific stimulatory action on the aromatizing enzyme system.

Estradiol-17beta biosynthesis in cultured granulosa cells from hypophysectomized immature rats; stimulation by follicle-stimulating hormone.

Granulosa cells isolated from the ovaries of hypophysectomized immature rats synthesize and secrete estradiol-17beta and estrone when grown for 2 days in monolayer culture in a synthetic medium containing testosterone (0.5 muM) and a highly purified follicle-stimulating hormone (FSH) preparation (0.25 mug/ml). Secretion is negligible in the absence of either testosterone or FSH, and a highly purified luteinizing hormone (LH) preparation (0.25 mug/ml) was without significant stimulatory effect. It is concluded that FSH regulates estrogen biosynthesis in granulosa cells of hypophysectomized rats by a specific stimulation of the aromatizing enzyme system.

Follicle-stimulating hormone induction of luteinizing hormone receptor in cultured rat granulosa cells: an examination of the need for steroids in the induction process.

The induction of LH receptors by FSH in cultured rat granulosa cells and the effects of ovarian steroids on this process were examined. Granulosa cells were isolated from the ovaries of untreated immature rats (25 days old) and cultured with highly purified FSH (Sairam; 250 ng/ml). After culture (48-96 h in chemically defined media), both the binding of [125I]hCG and the responsiveness (cAMP and progesterone production) to an acute LH stimulus (100 ng/ml; NIH B8) were measured. The appearance of LH/hCG-binding sites and LH responsiveness indicates the presence of functional LH receptors. The induction of LH receptors by FSH requires a lag period of 24-48 h. After 48 h, the concentration of LH receptors in cultured granulosa cells continues to increase with time in culture with FSH; the continuous presence of FSH is required to maintain the induction process. If granulosa cells are cultured without hormone for 24-48 h before FSH is added, no induction of LH receptors occurs. However, if 17 beta-estradiol (5 X 10(-7) M) is added during this initial period, then the cells are responsive to the later addition of FSH. This maintenance of FSH responsiveness is not observed when dihydrotestosterone or progesterone is substituted for 17 beta-estradiol in the initial culture period. The FSH-dependent induction of LH receptors in cultured rat granulosa cells can be blocked if an inhibitor of steroidogenesis, such as aminoglutethimide phosphate (AGP; 1 mM), is added along with FSH at the initiation of the culture. The inclusion of progesterone, dihydrotestosterone, or 17 beta-estradiol (but not 20 alpha-dihydroprogesterone) in the culture together with FSH and AGP will overcome the inhibition by AGP and restore the induction of LH receptors. The results suggest that steroids produced by the developing follicle can modulate the FSH-dependent induction of LH receptors, and this may play a role in the development of follicular responsiveness to LH.

Lack of ovarian responsiveness to gonadotropic hormones in infantile rats sterilized with busulfan.

Ovarian responsiveness to FSH and LH was examined in infantile rats treated in utero with busulfan (1,4-butanediol dimethanesulfate), a cytotoxic drug which has been shown to cause selective attrition of germ cells in the rat fetus. Pregnant Sprague-Dawley rats were injected ip on day 13 of gestation with busulfan (10 mg/kg BW) suspended in sesame oil or with sesame oil alone (control). Pups were killed on days 6, 8, 10, 12, or 14 postnatally, and trunk blood was collected. The ovaries were removed and either fixed for light microscopy or assessed for responsiveness to FSH and LH by their ability to produce net accumulations of cAMP and gonadal steroids in short term incubations. Ovaries, in which the germ cells were successfully destroyed, consisted of anastomotic cords of the intraovarian rete system surrounded by undifferentiated stromal tissue. A variable number of oocytes usually survived the busulfan treatment and were situated within the cords in irregularly defined follicles. Few treated oocytes proceeded to organize antral follicles by 14 days postnatally, but these follicles showed signs of normal theca and interstitial cell investment. A challenge of FSH or LH in vitro failed to stimulate net accumulations of cAMP, progesterone, androstenedione, or estradiol from treated ovaries whereas these responses were significantly stimulated in controls. Detectable levels of cAMP and steroids were, however, present in incubations of busulfan-treated ovaries on days 12 and 14, and these are likely attributable to the activity of antral follicles that survived the effects of busulfan. From day 8 to 12 plasma gonadotropin levels in treated animals rose significantly above those of controls suggesting that normal ovarian steroidogenesis is also suppressed in treated animals in vivo. Although direct effects of busulfan on somatic cells cannot be dismissed, these results suggest that the presence of germ cells is a prerequisite for the normal development of steroidogenic function in the rat ovary.

The in vivo and in vitro effects of exogenous leptin on ovulation in the rat.

Leptin, a hormonal product of the Lep gene, is expressed by adipocytes and is thought to play a role in regulating food intake and reproduction. The leptin protein has been localized in many reproductive tissues, including the ovary. Several publications indicate that the ovary is directly affected by leptin and that leptin may be a factor linking obesity and reproductive dysfunction. In this study, the effect of systemic leptin administration on ovulation in the rat ovary, both in vivo and in vitro, was investigated. Ip administration of leptin (30 microg at 3 hourly intervals for 15 h) to immature gonadotropin-primed rats caused a decline in ovulation in vivo, from 15.9+/-2.0 oocytes in the control animals to 5.3+/-1.6 oocytes in the leptin-treated animals (P < 0.001). Plasma progesterone and estradiol levels were analyzed immediately before ovulation, and neither was altered significantly in animals receiving the leptin treatment. Food consumption and body weight decreased following leptin treatment; however, a loss in body weight alone (pair-fed controls) was insufficient to explain the decrease in ovulation observed in the leptin-treated animals. In vitro perfusion of FSH-primed whole ovaries showed that treatment with leptin in combination with LH significantly decreased ovulations from 5.7+/-1.6 per ovary perfused with LH alone to 1.3+/-0.6 in those with LH and 1 microg/ml leptin (P < 0.05). Progesterone and estradiol levels in the samples taken during the perfusion period were unaffected by leptin treatment. In summary, leptin administration resulted in fewer ovulations, both in vivo and in vitro, but did not influence steroid levels. Systemic leptin administration at these doses can therefore inhibit ovulation, a process that occurs through a direct effect on the ovary.

Steroid biosynthesis by isolated human ovarian follicular cells in vitro.

Cultured thecal preparations from human ovarian follicles obtained from patients during the follicular phase of the menstrual cycle produced large amounts of delta 4-androstenedione and smaller amounts of testosterone and other androgens when stimulated by hCG, (Bu)2cAMP, and prostaglandin E2, but not when stimulated by FSH. Prostaglandin E2 also stimulated thecal cAMP production under the same conditions. By contrast, androgen production by granulosa cells was negligible with or without gonadotropins in the culture medium. Granulosa cells also did not produce 17 beta-estradiol, even when exposed to FSH and hCG, unless supplied with exogenous delta 4-androstenedione. On the other had, thecal, preparations did not produce significant amounts of 17 beta-estradiol even in the presence of FSH, hCG, and relatively large amounts of exogenous delta 4-androstenedione. These observations suggest that delta 4-androstenedione produced by LH or hCG-stimulated thecal cells is the main substrate for the synthesis of 17 beta-estradiol by FSH-stimulated granulosa cells in vivo.