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.

Influence of semen on inflammatory modulators of embryo implantation.

Insemination transmits to the female reproductive tract constituents of seminal plasma that target uterine epithelial cells to activate a cascade of inflammatory and immunological changes. Experiments in rodents show seminal factor signalling acts to 'condition' the female immune response to tolerate the conceptus, and to organise molecular and cellular changes in the endometrium to facilitate embryo development and implantation. The active factors in seminal plasma are identified as members of the transforming growth factor-beta family, with the relative balance of active moieties influencing the precise character of the female tract response. Experiments in rodents show that disruption of seminal plasma priming causes foetal growth retardation and changes in placental structure, with long-term consequences for the growth of the neonate. Recent studies indicate a similar physiological function and molecular basis for seminal plasma actions in the pig. In gilts, seminal plasma elicits an endometrial response characterised by recruitment of inflammatory leukocytes and induction of several pro-inflammatory cytokines and cyclo-oxygenase-2. The consequences are evident throughout the pre-implantation period of early pregnancy with altered leukocyte populations and cytokine parameters seen for at least 9 days. Exposure to semen also alters the dynamics in pre-implantation embryo development with an increase in the number of embryos and in their viability. Furthermore seminal plasma influences the temporal kinetics of ovulation, corpus luteum development and steroid production in the ovary. Dissecting the actions of seminal plasma may facilitate development of strategies to ensure maximal fertility and reduce embryo mortality in the pig.

Seminal plasma regulates ovarian progesterone production, leukocyte recruitment and follicular cell responses in the pig.

Seminal plasma (SP) acts to influence the uterine endometrium after mating, activating synthesis of embryotrophic cytokines and inflammatory changes that condition the tract for embryo implantation and establishing pregnancy. The objective of this study was to investigate in pigs whether the ovary might also be responsive to SP exposure. Prepubertal gilts were synchronised with exogenous gonadotrophins and received transcervical treatment with pooled boar SP or PBS; then the ovarian tissue was recovered at 34 h (preovulation) and on days 5 and 9 after treatment. The ovarian response was assessed by measuring ovulation rate, number and size of corpora lutea, ovarian leukocyte populations, progesterone production in vivo, as well as responses of retrieved granulosa cells cultured in vitro. In SP-treated gilts, leukocyte recruitment into the ovarian tissues was increased fourfold at 34 h, with macrophages comprising the most abundant cell lineage. There was no effect of SP on the number of oocytes ovulated; however, the weight of corpora lutea was increased in SP-treated gilts. SP also induced an increase in plasma progesterone content seen from day 5 to at least day 9 after treatment. In addition, granulosa cells and thecal tissue retrieved from preovulatory follicles of SP-treated gilts were more responsive in vitro to growth factor- and gonadotrophin-stimulated cell proliferation and progesterone synthesis. These results suggest that uterine exposure to SP influences immune cell trafficking in the ovary and enhances steroidogenesis in early pregnancy. The effects of SP on ovarian function potentially contribute to reproductive success in the pig.

Adipokines: implications for female fertility and obesity.

Obesity is associated with a diverse set of metabolic disorders, and has reproductive consequences that are complex and not well understood. The adipose tissue-produced leptin has dominated the literature with regards to female fertility complications, but it is pertinent to explore the likely role of other adipokines--adiponectin and resistin--as our understanding of their biological functions emerge. Leptin influences the developing embryo, the functioning of the ovary and the endometrium, interacts with the release and activity of gonadotrophins and the hormones that control their synthesis. In this review such biological actions and potential roles of the adipokines leptin, adiponectin and resistin are explored in relation to female fertility and the complexity of the obese metabolic state.

Androgens augment the mitogenic effects of oocyte-secreted factors and growth differentiation factor 9 on porcine granulosa cells.

In this study, we test the hypothesis that the growth-promoting action of androgens on granulosa cells requires paracrine signaling from the oocyte. Mural granulosa cells (MGCs) from small antral (1-3 mm) prepubertal pig follicles were cultured in the presence or absence of denuded oocytes (DO) from the same follicles to determine whether mitogenic and/or steroidogenic responses, to combinations of FSH, insulin-like growth factor 1 (IGF1), and dihydrotestosterone (DHT) were influenced by oocyte-secreted factors (OSFs). To further explore the identity of such factors we performed the same experiments, substituting growth differentiation factor 9 (GDF9), a known OSF, for the DO. OSFs and GDF9 both potently enhanced IGF1-stimulated proliferation, and inhibited FSH-stimulated progesterone secretion. Alone, DHT had little effect on DNA synthesis, but significantly enhanced the mitogenic effects of OSFs or GDF9 in the presence of IGF1. Denuded oocytes, GDF9, and DHT independently inhibited FSH-stimulated progesterone secretion, and androgen, together with DO or GDF9, caused the most potent steroidogenic inhibition. Focusing on mitogenic effects, we demonstrate that both natural androgen receptor (AR) agonists, testosterone and DHT, dose-dependently augmented the mitogenic activity of DO or GDF9. Antiandrogen (hydroxyflutamide) treatment, which is used to block androgen receptor activity, opposed the interaction between androgen and GDF9. In conclusion, androgens stimulate porcine MGC proliferation in vitro by potentiating the growth-promoting effects of oocytes or GDF9, via a mechanism that involves the AR. These signaling pathways are likely to be important regulators of folliculogenesis in vivo, and may contribute to the excess follicle growth that is observed in androgen-treated female animals.

Effects of recombinant human follicle-stimulating hormone on embryo development in mice.

We have developed a protocol using recombinant human follicle-stimulating hormone (rhFSH) to induce ovarian stimulation in the mouse to investigate its impact on preimplantation embryo development. Embryos were collected from adult female C57Bl/6 x CBA F1 mice treated with rhFSH (0, 2.5, 5.0, 10.0, or 20.0 IU) or 5 IU equine chorionic gonadotropin (eCG). Embryos were also recovered from nontreated control mice. Embryos were cultured in vitro for 88 h, and the stage of development was morphologically assessed. The allocation of cells to the inner cell mass or trophectoderm of blastocysts was determined by differential nuclear staining. The expression of insulin-like growth factor 2 (IGF-II), the insulin-like growth factor receptor (IGF-II receptor), and vascular endothelial growth factor (VEGF) in blastocysts was measured by real-time RT-PCR. Blastocyst development was reduced in the 10 (72.3 +/- 5.1%) and 20 (77.3 +/- 5.6%) IU rhFSH groups compared with control embryos (96.7 +/- 1.0%). The number of inner cell mass cells was reduced (P < 0.001) in the 5, 10, and 20 IU rhFSH groups and the eCG group compared with control embryos. We did not find any effect of rhFSH treatment on IGF-II, IGF-II receptor, or VEGF expression in blastocysts compared with the control group. eCG treatment, however, significantly increased the expression of IGF-II in blastocysts. These results indicate that ovarian stimulation with rhFSH impairs the in vitro development of preimplantation mouse embryos, and these results may have potential implications for clinical ovarian stimulation during infertility treatment and subsequent embryo quality.

Changes in ovarian, follicular, and oocyte morphology immediately after the onset of puberty are not accompanied by an increase in oocyte developmental competence in the pig.

This study was conducted to determine whether ovarian morphology and developmental competence of in vitro-matured (IVM) oocytes is immediately affected by the onset of puberty in the pig. Ovaries of peri-pubertal pigs were sorted into two groups according to the presence or absence of corpora lutea presence (CL and NCL, respectively. Ovary dimensions, follicle diameter and number, and oocyte diameter (with and without zona pellucidae) were determined. The developmental competence of in vitro-matured oocytes from these two groups was evaluated following parthenogenetic activation and culture in vitro. CL ovaries were significantly (P<0.01) larger than NCL ovaries (width: 22.3+/-0.9 mm versus 15.9+/-0.4 mm, length: 33.2+/-1 mm versus 24.1+/-0.4 mm). Although CL ovaries had fewer antral follicles in total compared with NCL ovaries (21.1+/-1.8 mm versus 46.8+/-2.2 mm), they had a similar number of follicles 3-8mm in diameter. The mean diameter of follicles that were aspirated was greater for CL ovaries than for NCL ovaries (4.5+/-0.1 mm versus 3.3+/-0.02 mm). Oocytes from CL ovaries were greater in diameter compared with those from NCL ovaries (zona retained: 159+/-1.3 microm versus 146.1+/-1.5 microm, zona free: 124.7+/-1.8 microm versus 113.1+/-1.6 microm). No differences were found between oocytes from CL and NCL ovaries for rates of meiotic maturation (91.6+/-3.2% versus 92.4+/-3.2%), cleavage (88.4+/-11% versus 90.7+/-2.6%) and blastocyst formation (21.0+/-3.7% versus 23.7+/-5.7%). Therefore, the onset of puberty coincides with immediate changes in ovarian morphology, increased ovary size, follicle and oocyte diameter, but not with improved oocyte developmental competence. This suggests that the higher developmental competence usually observed in adult oocytes is acquired gradually and requires exposure to multiple estrus cycles.

Oocyte-somatic cell interactions during follicle development in mammals.

Our current perspectives on the relationship between the oocyte and its surrounding somatic cells are changing as we gain a greater understanding of factors regulating folliculogenesis. It is now widely accepted that the oocyte plays a very active role in promoting follicle growth and directing granulosa cell differentiation. The oocyte achieves this, in part, by secreting soluble paracrine growth factors that act on its neighboring granulosa cells, which in turn regulate oocyte development. In preantral follicles, the oocyte directs granulosa cells to regulate oocyte growth, and oocytes may also directly drive follicle growth. In antral follicles, the oocyte governs the behaviour of cells in its immediate vicinity, thereby actively regulating its own microenvironment. As such, the oocyte establishes and maintains the distinct cumulus lineage of granulosa cells. This oocyte-cumulus cell interaction, in general, prevents luteinization of cumulus cells by promoting growth, regulating steroidogenesis and inhibin synthesis, and suppressing luteinizing hormone receptor expression. Conversely, mural granulosa cells in antral follicles, which have no direct physical contact with the oocyte and, presumably, experience a more diffuse concentration of oocyte-secreted factors, proceed to a different phenotype. In the ovulating follicle, oocyte-secreted factors also play vital roles in enabling cumulus cell expansion and regulating extracellular matrix stability, thus facilitating ovulation. The identities of these oocyte-secreted growth factors regulating such key ovarian functions remain unknown, although growth differentiation factor-9 (GDF-9), GDF-9B and/or bone morphogenetic protein-6 (BMP-6) are likely candidate molecules, probably forming complex local interactions with other related members of the transforming growth factor-beta (TGF-beta) superfamily. Elucidating the nature of oocyte-somatic cell interactions at the various stages of follicle development will have important implications for our understanding of factors regulating folliculogenesis, ovulation rate and fecundity.

Seminal plasma regulates endometrial cytokine expression, leukocyte recruitment and embryo development in the pig.

In pigs, uterine exposure to the constituents of semen is known to increase litter size but the underlying physiological mechanisms remain undefined. Studies in rodents and humans implicate immune modulating moieties in seminal plasma as likely candidates, acting through enhancing the receptivity of the female tract. In this study, the acute and longer term effects of seminal plasma on cytokine expression and leukocyte abundance in the pig endometrium during early pregnancy have been characterised. The reproductive tracts of gonadotrophin-primed pre-pubertal gilts treated with intrauterine infusions of either pooled seminal plasma or phosphate-buffered saline (PBS) were retrieved at 34 h, or on day 5 and day 9 after treatment. Seminal plasma elicited an endometrial inflammatory infiltrate comprised of predominantly macrophages and major histocompatibility complex class II+-activated macrophages and dendritic cells. The abundance of these cells was greatest at the pre-ovulatory (34 h) time-point and their increase relative to PBS-treated tissues was maintained until day 9 after seminal plasma treatment. Seminal plasma induced the expression of the cytokines, granulocyte macrophage colony-stimulating factor, interleukin-6 and monocyte chemoattractant protein-1, and the eicosanoid-synthesising enzyme cyclo-oxygenase-2. Expression was maximal 34 h after treatment but altered expression patterns as a consequence of seminal plasma induction persisted through early pregnancy. These changes were accompanied by altered dynamics in pre-implantation embryo development with an increase in the number of embryos and in their viability after seminal plasma treatment. Together, these findings implicate factors in seminal plasma in programming the trajectory of uterine cytokine expression and leukocyte trafficking during early pregnancy and in regulating pre-implantation embryo development in the pig.

Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity.

Compared with oocytes matured in vivo, in vitro-matured oocytes are compromised in their capacity to support early embryo development. Delaying spontaneous in vitro meiotic maturation using specific phosphodiesterase (PDE) isoenzyme inhibitors may permit more complete oocyte cytoplasmic maturation, possibly by prolonging cumulus cell (CC)-oocyte gap junctional communication during meiotic resumption. This study aimed to investigate the effect of the isoenzyme 3- (oocyte) and isoenzyme 4- (granulosa cell) specific PDE inhibitors on the kinetics of in vitro maturation and on subsequent oocyte developmental competence. Cumulus-oocyte complexes from antral bovine follicles were isolated and cultured in the presence of the specific PDE inhibitors milrinone (type 3) or rolipram (type 4) (100 microM). In the presence of FSH, both PDE inhibitors only slightly extended CC-oocyte gap junctional communication over the first 9 h, but they completely blocked meiotic resumption during this period (P < 0.001). The indefinite inhibitory effect of milrinone on meiotic resumption (30% at germinal vesicle stage after 48 h) was overridden by 24 h when treated with FSH, but not with hCG, suggesting a form of induced meiotic resumption. Oocytes treated with FSH with or without either PDE inhibitor were inseminated at either 24, 26, or 28 h. Treated with either the type 3 or type 4 PDE inhibitor significantly (P < 0.05) increased embryo development to the blastocyst stage by 33%-39% (to an average of 52% blastocysts) compared with control oocytes (38%) after insemination at 28 h, and significantly (P < 0.05) increased blastocyst cell numbers when inseminated at 24 h. These results suggest that delayed spontaneous meiotic maturation, coupled with extended gap junctional communication between the CCs and the oocyte has a positive effect on oocyte cytoplasmic maturation, thereby improving oocyte developmental potential.

Oxygen-regulated gene expression in bovine blastocysts.

Oxygen concentrations used during in vitro embryo culture can influence embryo development, cell numbers, and gene expression. Here we propose that the preimplantation bovine embryo possesses a molecular mechanism for the detection of, and response to, oxygen, mediated by a family of basic helix-loop-helix transcription factors, the hypoxia-inducible factors (HIFs). Day 5 compacting bovine embryos were cultured under different oxygen tensions (2%, 7%, 20%) and the effect on the expression of oxygen-regulated genes, development, and cell number allocation and HIFalpha protein localization were examined. Bovine in vitro-produced embryos responded to variations in oxygen concentration by altering gene expression. GLUT1 expression was higher following 2% oxygen culture compared with 7% and 20% cultured blastocysts. HIF mRNA expression (HIF1alpha, HIF2alpha) was unaltered by oxygen concentration. HIF2alpha protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1alpha protein was undetectable at any oxygen concentration or in the presence of the HIF protein stabilizer desferrioxamine (DFO), despite being detectable in cumulus cells following normal maturation conditions, acute anoxic culture, or in the presence of DFO. Oxygen concentration also significantly altered inner cell mass cell proportions at the blastocyst stage. These results suggest that oxygen can influence gene expression in the bovine embryo during postcompaction development and that these effects may be mediated by HIF2alpha.

Interactions between androgen and growth factors in granulosa cell subtypes of porcine antral follicles.

Androgens acting via the androgen receptor (AR) have been implicated in regulation of folliculogenesis in many animal species. These effects are possibly mediated via enhancement of FSH and/or insulin-like growth factor (IGF)-I activity in granulosa cells, which contain high levels of AR protein. We examined the in vitro effect of dihydrotestosterone (DHT) on DNA synthesis and progesterone secretion by follicular cells in response to FSH and IGF-I, alone or in combination. Cells from separate pools of 1- to 3-mm and 3- to 5-mm antral follicles were aspirated from gilt ovaries and fractioned into mural granulosa cells (MGCs) and cumulus-oocyte complexes (COCs) for subsequent cell culture. Androgen alone or with any combination of mitogen had minimal effect on proliferative and no effect on steroidogenic responses of MGCs from 3- to 5-mm antral follicles. Conversely, in MGCs from 1- to 3-mm follicles, DHT significantly enhanced IFG-I-stimulated proliferation and had variable influence on progesterone secretion. The effects of DHT on proliferative responses of COCs were also dependent on follicle size: DHT significantly augmented either IGF-I-stimulated proliferation (1- to 3-mm follicles) or FSH-stimulated proliferation (3- to 5-mm follicles). However, the steroidogenic responses of all COCs were identical, whereby DHT significantly suppressed progesterone secretion, predominantly in the presence of FSH. Addition of an AR antagonist, hydroxyflutamide, generally reversed the proliferative responses invoked by DHT but not the steroidogenic responses. We conclude that androgen-receptor-mediated activity in granulosa cells of antral follicles is dependent on follicle size, is influenced by proximity of cells to the oocyte, and possibly involves both classic and nonclassic steroid mechanisms.

Comparison of oocyte factors and transforming growth factor-beta in the regulation of DNA synthesis in bovine granulosa cells.

Oocytes are powerful local modulators of follicular cell functions and many of the activities of oocytes are attributed to members of the transforming growth factor-beta (TGF-beta) superfamily. Whilst in the mouse it is known that members of this family are able to mimic many of the effects of oocytes on follicular cells, the relative importance of any of these factors is unknown in bovine follicles. The objectives of this study were to determine if bovine oocytes express and secrete TGF-beta and to compare oocyte-secreted factor(s) to TGF-beta in terms of their capacities to stimulate mural granulosa cell (MGC) DNA synthesis. Bovine ovaries were collected from an abattoir and RNA was extracted from isolated MGC, cumulus cells, cumulus-oocyte complexes and denuded oocytes (DO). Using RT-PCR, all cell types were found to express TGF-beta1 and TGF-beta2 mRNA transcripts. However, no TGF-beta bioactivity could be detected from DO using a sensitive (40 pg/ml) and specific mink lung fibroblast cell bioassay. MGC were cultured with various combinations and doses of TGF-beta2 and DO for 18 h, followed by a 6-h pulse of [3H]-thymidine as an indicator of cellular DNA synthesis. MGC DNA synthesis was stimulated by both TGF-beta2 and DO. However in response to increasing doses of TGF-beta2, [3H]-thymidine levels plateaued at <2-fold above control levels, whereas levels continued to increase over the dose range of DO tested (up to 3.4-fold). Addition of a TGF-beta pan-specific neutralising antibody to MGC cultures eliminated the TGF-beta2-stimulatory effects on DNA synthesis and the TGF-beta2-suppressive effects on progesterone production, but the antibody was unable to neutralise the same responses when induced by DO. These results support a role for TGF-beta1, TGF-beta2 and DO in paracrine/autocrine regulation of bovine granulosa cell function, but indicate that neither TGF-beta1 nor TGF-beta2 can account for the actions of bovine oocytes on granulosa cells.

Effects of leptin administration and feed restriction on thecal leucocytes in the preovulatory rat ovary and the effects of leptin on meiotic maturation, granulosa cell proliferation, steroid hormone and PGE2 release in cultured rat ovarian follicles.

Leptin is expressed by adipocytes and is thought to play a role in regulating food intake and in reproduction. It has been demonstrated that acute leptin administration to immature gonadotrophin-primed rats in vivo inhibits ovulation and causes a decline in food intake. However, feed restriction alone does not inhibit ovulation. Two experiments were designed to investigate the mechanism of leptin-induced inhibition of ovulation. In the first experiment, which was prompted by the importance of ovarian leucocytes in ovulation, the role of leucocytes in leptin-induced inhibition of ovulation was investigated. The second experiment investigated whether high leptin concentrations could inhibit other factors important to ovulation, such as meiotic competence of oocytes, granulosa cell proliferation, steroid or PGE(2) release, and interleukin 1beta production, in vitro. In the first experiment, the populations of neutrophils and monocytes-macrophages in the preovulatory follicles of gonadotrophin-primed, leptin-treated and -untreated rats were examined. A decrease in food intake, as a result of either leptin treatment or feed restriction, specifically reduced the numbers of neutrophils and monocytes-macrophages infiltrating the theca interna of preovulatory follicles without affecting the numbers found in the stroma. The findings show that reduced infiltration of thecal neutrophils and macrophages into preovulatory follicles is a response to reduced food intake. Furthermore, this reduction is not the direct cause of the leptin-induced inhibition of ovulation. In the second experiment, ovarian follicles were cultured for 4 or 12 h in the presence or absence of the following hormones: FSH (500 miu), insulin-like growth factor I (IGF-I) (50 ng ml(-1)), LH (100 ng ml(-1)) and leptin (300 ng ml(-1)). The results demonstrated that high concentrations of leptin in follicle culture do not affect meiotic maturation or steroid release, but tend to inhibit release of PGE 2 (although this result was not significant). DNA synthesis in granulosa cells was not inhibited by leptin in FSH- and IGF-I-supplemented culture media. These results are in agreement with previous studies that have shown that leptin inhibits the stimulatory effects of IGF-I on FSH-stimulated oestradiol production in rat granulosa cells without affecting progesterone production. In summary, leptin does not appear to have an adverse effect on the components of ovulation tested in this study, and therefore must impact on the ovulatory cascade in a way that remains to be defined.

Effects of maternal age on oocyte developmental competence.

The widespread use of a variety of assisted reproductive technologies has removed many of the constraints that previously restricted mammalian reproduction to the period between onset of puberty and reproductive senescence. In vitro embryo production systems now allow oocytes from very young animals to undergo fertilization and form embryos capable of development to normal offspring, albeit at somewhat reduced efficiencies compared to oocytes from adult females. They also can overcome infertility associated with advanced age of animals and women. This review examines oocyte developmental competence as the limiting factor in applications of assisted reproductive technologies for both juvenile and aged females. Age of oocyte donor is a significant factor influencing developmental competence of the oocyte. Age-related abnormalities of oocytes include a) meiotic incompetence or inability to complete meiotic maturation resulting in oocytes incapable of fertilization; b) errors in meiosis that can be compatible with fertilization but lead to genetic abnormalities that compromise embryo viability; and c) cytoplasmic deficiencies that are expressed at several stages of development before or after fertilization. In general, oocytes from juvenile donors and the embryos derived therefrom appear less robust and may be less tolerant to suboptimal handling and in vitro culture conditions than are adult oocytes. Research to identify specific cytoplasmic deficiencies of juvenile oocytes may enable modifications of culture conditions to correct such deficiencies and thus enhance developmental competence. Use of oocytes from aged donors for assisted reproduction can have a variety of applications such as extending the reproductive life of individual old females whose offspring still have high commercial value, and conservation of genetic resources such as rare breeds of livestock and endangered species. In general, female fertility decreases with advancing age. Studies of women in oocyte donation programs have established reduced oocyte competence as the major cause of declining fertility with age, although inadequate endometrial function can also be a contributing factor. Most research has emphasized the importance of chromosomal abnormalities because of the well established increase in aneuploidy with increasing maternal age but little is known about the underlying cellular and molecular mechanisms. Research aimed at identifying the specific developmental deficiencies of oocytes from juvenile donors and abnormalities of oocytes from aged females will assist in overcoming present bottlenecks that limit the efficiency of assisted reproduction technologies. Such research will also be crucial to the development of new oocyte-based technologies for overcoming infertility and possibly subverting chromosomal abnormalities in women approaching menopause.

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.

Mouse oocyte mitogenic activity is developmentally coordinated throughout folliculogenesis and meiotic maturation.

Oocytes secrete soluble factors that regulate the growth and differentiation of follicular cells, including maintenance of the distinctive cumulus cell phenotype. This study determines whether the mitogenic activity of oocytes is developmentally regulated and examines the responsiveness of follicular cells to oocytes at different stages of follicular development. Prepubertal SV129 mice of varying ages were primed with 5 IU equine chorionic gonadotropin (eCG) and oocytes/zygotes collected either 46 h post-eCG (immature oocytes), 12 h after administration of 5 IU human CG (hCG; ovulated ova), or 12 h post-hCG and mating (zygotes). Mural granulosa cells (MGC) from antral follicles and GC from preantral follicles were cultured +/- denuded oocytes (DO) for 18 h, followed by a 6-h pulse of [(3)H]thymidine as an indicator of cellular DNA synthesis. Coculturing MGC with meiotically maturing oocytes led to a dose-dependent increase in [(3)H]thymidine incorporation (20-fold above control levels at 0.5 DO/microl). However, [(3)H] counts remained unchanged from control levels when cultured with meiotically incompetent DO from 11- to 15-day-old mice (3% germinal vesicle breakdown; GVB), irrespective of dose of DO or developmental status of GC (MGC or preantral GC). In some treatments, spontaneous meiotic resumption of competent oocytes was prevented by culturing with 5 microM milrinone, a selective inhibitor of oocyte-specific cyclic nucleotide phosphodiesterase. The mitogenic capacity of oocytes was found to decline during and after oocyte maturation. [(3)H]Thymidine incorporation in MGC was highest (11-fold above controls) when cultured with meiotically inhibited (milrinone-treated) GV DO, stimulated 5.5-fold by culture with maturing oocytes, 3-fold with ovulated ova, and unstimulated by zygotes. [(3)H]Thymidine incorporation in MGC was not altered by the dose of milrinone, either in the presence or absence of DO. Metaphase I marked the beginning of the decline in the capacity of oocytes to promote MGC DNA synthesis. These results demonstrate that the capacity of oocytes to promote proliferation of granulosa cells follows a developmental program, closely linked to oocyte meiotic status, increasing with the acquisition of meiotic competence and declining during and after oocyte maturation.

Effect of cell-to-cell contact on in vitro deoxyribonucleic acid synthesis and apoptosis responses of bovine granulosa cells to insulin-like growth factor-I and epidermal growth factor.

Follicle development is the result of a balanced ratio between cell proliferation and cell death. Previous studies demonstrated differential mitotic responses to insulin-like growth factor (IGF)-I and epidermal growth factor (EGF) of cumulus cells (CC) and mural granulosa cells (MGC). Because cell-to-cell contact seems to modulate the occurrence of programmed cell death, the present experiments investigated the role of cell association in mediating apoptosis and the mitogenic responses to these growth factors of CC and MGC. Cumulus cells were cultured either as intact cumulus-oocyte complexes (COC) or after dissociation with EGTA + sucrose, in the presence of 50 ng/ml IGF-I, 5 ng/ml EGF, or both. Mural granulosa cells from the same follicles were similarly cultured either as cell aggregates or as dissociated cells. Synthesis of DNA was assessed by measurement of [(3)H]thymidine incorporation during the last 6 h of a 24-h culture in TCM199. Percentages of cells undergoing apoptosis were determined immunohistochemically in intact COC and GC aggregates, before and after dissociation as well as after the culture period. Epidermal growth factor and IGF-I stimulated DNA synthesis in both cell types; however, EGF inhibited the action of IGF-I in intact COC but not in MGC. Compared to nondissociated cells, dissociation resulted in a reduction of the mitogenic response of CC to both growth factors and of MGC to EGF. Unlike the response of intact COC to combined treatment with the two growth factors, dissociated CC displayed additive responses to the two growth factors in combination. Addition of denuded oocytes to cultures of dissociated CC enhanced both basal and growth factor-stimulated DNA synthesis but did not restore the inhibitory effect of EGF on the IGF-I response characteristic of intact COC. A significant proportion of intact MGC aggregates underwent apoptosis after 24 h of culture, while no increase of apoptotic cells was observed in intact COC. A dramatic increase in the percentage of apoptotic cells was observed in both CC and MGC when cell-cell contact was interrupted, and EGF and IGF-I were able to partially prevent its occurrence. Taken together these data showed that CC and MGC exhibit qualitatively and quantitatively different responses to IGF-I when cultured in the presence of EGF both in terms of DNA synthesis and onset of apoptosis. Moreover, the disruption of cell-cell contact was a major factor reducing cell proliferation and inducing apoptosis among both subsets of GC.