Haemoglobin expression in in vivo murine preimplantation embryos suggests a role in oxygen-regulated gene expression.

Haemoglobin expression is not restricted to erythroid cells. We investigated the gene expression of the haemoglobin subunits haemoglobin, alpha adult chain 1 (Hba-a1) and haemoglobin, beta (Hbb), 2,3-bisphosphoglycerate mutase (Bpgm) and the oxygen-regulated genes BCL2/adenovirus E1B interacting protein 3 (Bnip3), solute carrier family 2 (facilitated glucose transporter), member 1 (Slc2a1) and N-myc downstream regulated gene 1 (Ndrg1) in the murine preimplantation embryo, comparing invivo to invitro gene expression. Relatively high levels of Hba-a1 and Hbb were expressed invivo from the 2-cell to blastocyst stage; in contrast, little or no expression occurred invitro. We hypothesised that the presence of haemoglobin invivo creates a low oxygen environment to induce oxygen-regulated gene expression, supported by high expression of Slc2a1 and Ndrg1 in invivo relative to invitro embryos. In addition, analysis of an invitro-derived human embryo gene expression public dataset revealed low expression of haemoglobin subunit alpha (HBA) and HBB, and high expression of BPGM. To explore whether there was a developmental stage-specific effect of haemoglobin, we added exogenous haemoglobin either up to the 4-cell stage or throughout development to the blastocyst stage, but observed no difference in blastocyst rate or the inner cell mass to trophectoderm cell ratio. We conclude that haemoglobin in the invivo preimplantation embryo raises an interesting premise of potential mechanisms for oxygen regulation, which may influence oxygen-regulated gene expression.

Effects of differing oocyte-secreted factors during mouse in vitro maturation on subsequent embryo and fetal development.

PURPOSE: We hypothesised that varying native oocyte-secreted factor (OSF) exposure or using different recombinant OSF peptides would have differential effects on post-in vitro maturation (IVM) embryo and fetal development. METHODS: Mouse cumulus oocyte complexes (COCs) were treated with the purified mature domain of GDF9 and/or BMP15 or were co-cultured with denuded oocytes (DOs) from 0 h or 3 h of IVM. DOs were matured for 3 h as either intact COCs+/-FSH before denuding, or as DOs + FSH. COCs were fertilised and blastocyst development was assessed on days 5 and 6, and either differentially stained for ICM numbers or vitrified/warmed embryos were transferred to recipients to assess implantation and fetal rates. RESULTS: No improvement in embryo development was observed with the addition of GDF9 and/or BMP15 to IVM. In contrast, embryos derived from COCs co-cultured with DOs had significantly improved blastocyst rates and ICM numbers compared to controls (P < 0.05). The highest response was obtained when DOs were first added to COCs at 3 h of IVM, after being pre-treated (0-3 h) as COCs + FSH. Compared to control, co-culture with DOs from 3 h did not affect implantation rates but more than doubled fetal yield (21% vs 48%; P < 0.05). GDF9 Western blot analysis was unable to detect any differences in quantity or form of GDF9 (17 and 65 kDa) in extracts of DO at 0 h or 3 h. CONCLUSIONS: This study provides new knowledge on means to improve oocyte quality in vitro which has the potential to significantly aid human infertility treatment and animal embryo production technologies.

Mode of oocyte maturation affects EGF-like peptide function and oocyte competence.

The function and impact of epidermal growth factor (EGF)-like peptide signalling during ovulation and in vivo oocyte maturation (IVV) has been recently characterized, however, little is currently known about the effect of oocyte in vitro maturation (IVM) on this pathway. The aim of this study was to examine expression and functional aspects of three EGF-like peptides (amphiregulin, epiregulin and betacellulin) and their common receptor (EGFR) in cumulus cells during mouse oocyte IVM compared with IVV. Cumulus-oocyte complexes (COCs) were collected from prepubertal mice either 46 h post-eCG (IVM) or 46 h post-eCG plus 0.5-12 h post-hCG (IVV). Time course experiments showed mRNA expression of all three EGF-like peptides and amphiregulin protein in IVM media were significantly lower for the majority of FSH-supplemented IVM compared with IVV. The supplementation of EGF during IVM yielded EGF-like peptide expression levels comparable with IVV and amphiregulin/epiregulin supplemented IVM. However, despite this, EGF activation of the COC EGFR remained significantly lower at 3 and 6 h of IVM than in vivo, and levels were similar to those observed during FSH-supplemented IVM. The addition of exogenous epiregulin during IVM significantly increased blastocyst rates, and epiregulin and amphiregulin improved blastocyst quality, compared with FSH or EGF. In conclusion, findings from this study suggest that the widely used IVM additives, FSH and EGF, are inadequate propagators of the essential EGF-like peptide signalling cascade. In contrast, the use of epiregulin and/or amphiregulin during IVM leads to improved oocyte developmental competence and therefore may be preferable IVM additives than FSH or EGF.

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.

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.

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.

Immunoneutralization of growth differentiation factor 9 reveals it partially accounts for mouse oocyte mitogenic activity.

Paracrine factors secreted by oocytes play a pivotal role in promoting early ovarian follicle growth and in defining a morphogenic gradient in antral follicles, yet the exact identities of these oocyte factors remain unknown. This study was conducted to determine the extent to which the mitogenic activity of mouse oocytes can be attributed to growth differentiation factor 9 (GDF9). To do this, specific anti-human GDF9 monoclonal antibodies were generated. Based on epitope mapping and bioassays, a GDF9 neutralizing antibody, mAb-GDF9-53, was characterized with very low cross-reactivity with related transforming growth factor (TGF)beta superfamily members, including BMP15 (also called GDF9B). Pep-SPOT epitope mapping showed that mAb-GDF9-53 recognizes a short 4-aa sequence, and three-dimensional peptide modeling suggested that this binding motif lies at the C-terminal fingertip of mGDF9. As predicted by sequence alignments and modeling, the antibody detected recombinant GDF9, but not BMP15 in a Western blot and GDF9 protein in oocyte extract and oocyte-conditioned medium. In a mouse mural granulosa cell (MGC) bioassay, mAb-GDF9-53 completely abolished the mitogenic effects of GDF9, but had no effect on TGFbeta1 or activin A-stimulated MGC proliferation. An unrelated IgG at the same dose had no effect on GDF9 activity. This GDF9 neutralizing antibody was then tested in an established oocyte-secreted mitogen bioassay, where denuded oocytes cocultured with granulosa cells promote cell proliferation in a dose-dependent manner. The mAb-GDF9-53 dose dependently (0-160 microg/ml) decreased the mitogenic activity of oocytes but only by approximately 45% at the maximum dose of mAb. Just 5 microg/ml of mAb-GDF9-53 neutralized 90% of recombinant mGDF9 mitogenic activity, but only 15% of oocyte activity. Unlike mAb-GDF9-53, a TGFbeta pan-specific neutralizing antibody did not affect the mitogenic capacity of the oocyte, but completely neutralized TGF beta 1-induced DNA synthesis. This study has characterized a specific GDF9 neutralizing antibody. Our data provide the first direct evidence that the endogenous GDF9 protein is an important oocyte-secreted mitogen, but also show that GDF9 accounts for only part of total oocyte bioactivity.

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.

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 granulocyte-macrophage colony-stimulating factor deficiency on ovarian follicular cell function.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine secreted by lymphohaemopoietic and other cell lineages, is known to influence ovarian cyclicity and embryo development. The aim of this study was to examine the effect of GM-CSF on ovarian follicular cell function using GM-CSF-deficient (GM -/-) mice. Immature GM -/- and GM +/+ mice were stimulated with eCG, and cumulus-oocyte complexes and mural granulosa cells were collected 48 h later. Expression of GM-CSF receptor (GM-CSFR) alpha and beta mRNA subunits by cumulus-oocyte complexes and mural granulosa cells was examined using RT-PCR. Cumulus-oocyte complexes from both genotypes were found to express mRNA for the GM-CSFRalpha-subunit only, while the mural granulosa cells expressed both the alpha and beta receptor subunits. Cumulus-oocyte complexes recovered from GM -/- mice had approximately twice the number of cumulus cells per cumulus-oocyte complex than did those of GM +/+ mice (P < 0.05), even though the growth-promoting activity of denuded GM -/- oocytes was found to be equivalent to that of wild-type oocytes. GM-CSF deficiency was associated with marginally increased DNA synthesis in cumulus cells and significantly (P < 0.05) lower progesterone production by mural granulosa cells recovered from GM -/- compared with those recovered from GM +/+ mice. The addition of rec-mGM-CSF in vitro did not affect DNA synthesis in either cell type or progesterone production by mural granulosa cells, irrespective of GM-CSF status. There was no effect of GM-CSF deficiency on the capacity of FSH and insulin-like growth factor I to stimulate DNA synthesis in cumulus-oocyte complexes (approximately 15- and threefold, respectively) and in mural granulosa cells (approximately two- and threefold, respectively). Taken together, these data show that GM-CSF influences events associated with follicular maturation in mice. The effects of GM-CSF are not exerted directly in granulosa or cumulus cells, but appear to be mediated indirectly, perhaps through the agency of steroidogenesis-regulating secretions of local macrophage populations residing in the theca.

Glomerular prostaglandin formation in two-kidney, one-clip hypertensive rats.

In vitro prostaglandin (PG) and thromboxane B2 (TXB2) formation by isolated glomeruli from normotensive (N) and two-kidney, one-clip hypertensive (2K,1C) rats was determined. When calculated on the basis of glomerular protein content, PGE2, 6-keto-PGF1 alpha and TXB2 production of glomeruli from clipped kidneys was significantly greater than PG and TXB2 formation of glomeruli from the untouched kidneys. When PG and TXB2 formation was calculated per amount of glomeruli, only PGE2 formation was found to be significantly greater in clipped kidneys. No severe damage of glomerular structure was found in the kidneys when studied by light microscopy. In additional in vivo studies, the effect of the cyclooxygenase inhibitor indomethacin on blood pressure and glomerular filtration rate (GFR) was evaluated. Following indomethacin GFR in 7 of 13 clipped kidneys of 2K,1C rats decreased from 363 +/- 77 to 188 +/- 51 microliter/100 g body wt, whereas six kidneys developed anuria. No effect of cyclooxygenase inhibition on GFR was found in N rats and in untouched kidneys of 2K,1C rats. Mean arterial blood pressure in 2K,1C hypertension fell significantly, from 158 +/- 10 to 135 +/- 7 mmHg, after cyclooxygenase inhibition. No effect was seen in N rats. The data suggest that increased glomerular PG formation in the clipped kidneys of 2K,1C rats is involved in the pathogenesis of hypertension in this animal model.