Gonadotropin elevation is ootoxic to ovulatory oocytes and inhibits oocyte maturation, and activin decoy receptor ActRIIB:Fc therapeutically restores maturation.

BACKGROUND: Elevated FSH often occurs in women of advanced maternal age (AMA, age ≥ 35) and in infertility patients undergoing controlled ovarian stimulation (COS). There is controversy on whether high endogenous FSH contributes to infertility and whether high exogenous FSH adversely impacts patient pregnancy rates. METHODS: The senescence-accelerated mouse-prone-8 (SAMP8) model of female reproductive aging was employed to assess the separate impacts of age and high FSH activity on the percentages (%) of viable and mature ovulated oocytes recovered after gonadotropin treatment. Young and midlife mice were treated with the FSH analog equine chorionic gonadotropin (eCG) to model both endogenous FSH elevation and exogenous FSH elevation. Previously we showed the activin inhibitor ActRIIB:Fc increases oocyte quality by preventing chromosome and spindle misalignments. Therefore, ActRIIB:Fc treatment was performed in an effort to increase % oocyte viability and % oocyte maturation. RESULTS: The high FSH activity of eCG is ootoxic to ovulatory oocytes, with greater decreases in % viable oocytes in midlife than young mice. High FSH activity of eCG potently inhibits oocyte maturation, decreasing the % of mature oocytes to similar degrees in young and midlife mice. ActRIIB:Fc treatment does not prevent eCG ootoxicity, but it restores most oocyte maturation impeded by eCG. CONCLUSIONS: FSH ootoxicity to ovulatory oocytes and FSH maturation inhibition pose a paradox given the well-known pro-growth and pro-maturation activities of FSH in the earlier stages of oocyte growth. We propose the FOOT Hypothesis ("FSH OoToxicity Hypothesis), that FSH ootoxicity to ovulatory oocytes comprises a new driver of infertility and low pregnancy success rates in DOR women attempting spontaneous pregnancy and in COS/IUI patients, especially AMA women. We speculate that endogenous FSH elevation also contributes to reduced fecundity in these DOR and COS/IUI patients. Restoration of oocyte maturation by ActRIB:Fc suggests that activin suppresses oocyte maturation in vivo. This contrasts with prior studies showing activin A promotes oocyte maturation in vitro. Improved oocyte maturation with agents that decrease endogenous activin activity with high specificity may have therapeutic benefit for COS/IVF patients, COS/IUI patients, and DOR patients attempting spontaneous pregnancies.

Maternal age and gonadotrophin elevation cooperatively decrease viable ovulated oocytes and increase ootoxicity, chromosome-, and spindle-misalignments: '2-Hit' and 'FSH-OoToxicity' mechanisms as new reproductive aging hypotheses.

While there is consensus that advanced maternal age (AMA) reduces oocyte yield and quality, the notion that high FSH reduces oocyte quality and causes aneuploidy remains controversial, perhaps due to difficulties controlling the confounding variables of age and FSH levels. Here, contributions of age and gonadotrophin elevation were separately controlled using a mouse model of human female reproductive aging. Ovulated oocytes were collected from young and midlife mice after 0-, 2.6-, or 17-day treatment with the FSH analog equine chorionic gonadotrophin (eCG), to model both exogenous FSH elevation within a single treatment cycle (as in controlled ovarian stimulation (COS)), and chronic endogenous FSH elevation during multiple cycles (as in diminished ovarian reserve). After 17-day eCG, fewer total oocytes/mouse are ovulated in midlife than young mice, and a precipitous decline in viable oocytes/mouse is observed in midlife but not young mice throughout eCG treatment. eCG is potently ootoxic to ovulatory oocytes and strongly induces chromosome- and spindle-misalignments within 2.6 days of eCG in midlife, but only after 17 days in young mice. These data indicate that AMA increases susceptibility to multiple adverse effects of elevated FSH activity in ovulated oocytes, including declines in total and viable oocytes/mouse, and induction of ootoxicity and aneuploidy. Two hypotheses are proposed for underlying causes of infertility in women. The FSH OOToxicity Hypothesis ('FOOT Hypothesis') posits that high FSH is ootoxic to ovulatory oocytes and that FSH ootoxicity is a root cause of low pregnancy success rates in naturally cycling women with high FSH and IUI patients undergoing COS. The '2-Hit Hypothesis' posits that AMA increases susceptibility to FSH-induced ootoxicity and aneuploidy.

Adiposity and weight gain during pregnancy associate independently with behavior of infant rhesus monkeys (Macaca mulatta).

Growing evidence identifies maternal adiposity as a potentially modifiable risk factor for adverse neurodevelopment. This retrospective cohort analysis examined whether maternal prepregnancy adiposity and gestational weight gain were associated with behavioral outcomes in 173 rhesus macaque infants at the California National Primate Research Center. Dams conceived indoors, had uncomplicated pregnancies, delivered vaginally, and reared infants indoors. Infants underwent standardized biobehavioral analysis at 90-120 days of age from 3/2001-5/2015. Offspring of mothers with greater baseline adiposity or gestational weight gain exhibited a pattern of poor adaptability characterized by greater emotionality as the assessments proceeded, blunted affective response to a human intruder challenge, and reduced interest in novel stimuli which is associated with poorer social functioning later in life. They also had lower cortisol levels following dexamethasone suppression, perhaps a response to cortisol excess during gestation. These results amplify growing public health concerns implicating maternal adiposity in impaired fetal neurobehavioral programming.

EGF-like ligands mediate progesterone's anti-apoptotic action on macaque granulosa cells.

A local autocrine/paracrine role for progesterone is an absolute requirement for corpus luteum formation in primates. Despite this, the mechanism(s) remain obscure, although existing data suggest an anti-apoptotic action to be central. There are a limited number of progestin-regulated gene targets identified in the luteinizing primate follicle, suggesting that a small number of important genes may mediate progesterone action. Possible gene targets could be the epidermal growth factor (EGF) family members amphiregulin (AREG) and epiregulin (EREG). Using macaques undergoing controlled ovarian stimulation cycles, we show that the phosphorylation of EGF receptor (EGFR), ERK 1/2, and AKT increases 6 h after an ovulatory human chorionic gonadotropin (hCG) stimulus and remains activate through 24 h. Immunoreactive EREG and AREG ligands in the follicular fluid both increased in a time frame commensurate with EGFR phosphorylation. The mRNA expression of AREG and EREG in nonluteinized granulosa cells (NLGC) was induced in culture with hCG, an effect blocked by progesterone receptor (PGR) antagonists. Overexpression of PGR B in NLGC and treatment with a nonmetabolizable progestin did not increase either gene, indicating both progesterone and luteinizing hormone/CG are necessary. Addition of EGF and EGF-like ligands did not promote steroidogenesis in vitro by granulosa cells in the presence of gonadotropin, but were able to partially reverse RU486-induced cell death. These data suggest that progesterone promotes the expression of AREG and EREG, which in turn maintain viability of luteinizing granulosa cells, representing one possible mechanism whereby progesterone promotes corpus luteum formation in the primate.

Ethanol, acetaldehyde, and estradiol affect growth and differentiation of rhesus monkey embryonic stem cells.

BACKGROUND: The timing of the origins of fetal alcohol syndrome has been difficult to determine, in part because of the challenge associated with in vivo studies of the peri-implantation stage of embryonic development. Because embryonic stem cells (ESCs) are derived from blastocyst stage embryos, they are used as a model for early embryo development. METHODS: Rhesus monkey ESC lines (ORMES-6 and ORMES-7) were treated with 0, 0.01, 0.1, or 1.0% ethanol, 1.0% ethanol with estradiol, or 0.00025% acetaldehyde with or without estradiol for 4 weeks. RESULTS: Although control ESCs remained unchanged, abnormal morphology of ESCs in the ethanol and acetaldehyde treatment groups was observed before 2 weeks of treatment. Immunofluorescence staining of key pluripotency markers (TRA-1-81 and alkaline phosphatase) indicated a loss of ESC pluripotency in the 1.0% ethanol group. ORMES-7 was more sensitive to effects of ethanol than ORMES-6. CONCLUSIONS: Estradiol appeared to increase sensitivity to ethanol in the ORMES-6 and ORMES-7 cell line. The morphological changes and labeling for pluripotency, proliferation, and apoptosis demonstrated that how ethanol affects these early cells that develop in culture, their differentiation state in particular. The effects of ethanol may be mediated in part through metabolic pathways regulating acetaldehyde formation, and while potentially accentuated by estradiol in some individuals, how remains to be determined.

Granulosa cell expression of G1/S phase cyclins and cyclin-dependent kinases in PMSG-induced follicle growth.

Follicular development involves a complex orchestration of granulosa cell proliferation and differentiation. It is becoming increasingly apparent that the rate of granulosa cell proliferation declines as follicles reach the large antral status, prior to an ovulatory gonadotropin stimulus, although a precise time course and mechanism for this decline has not been described. The goal of the present study was to characterize granulosa cell proliferation following the onset of antral follicle growth in PMSG-primed immature rats, with emphasis on G1/S phase cyclins and cyclin-dependent kinases. Flow cytometric analysis demonstrated that the percentage of granulosa cells in S phase peaked 24-30 h post-PMSG and declined to control levels 48 h after PMSG administration. Expression of both Cyclin D2 and Cdk 4 was highest 12h post-PMSG and decreased to control levels by 48 h. In addition, Cdk 2 protein increased transiently 12-24h after PMSG. Cyclin E expression increased significantly by 12h but remained elevated through 48 h, and multiple isoforms of Cyclin E were observed with increased proliferation. Both Cdk 4 and Cdk 2 activity parallel protein expression, although, changes in Cdk 2 were more marked. Levels of mRNA for the cell cycle inhibitors p21CIP1 and p27KIP1 increased significantly by 48 h post-PMSG. These results demonstrate that PMSG-stimulated movement of granulosa cells across the G1/S boundary during follicle growth is transient. In addition, the control of granulosa cell proliferation may reside through the regulation of both Cdk 2 and Cdk 4.

Activin Decoy Receptor ActRIIB:Fc Lowers FSH and Therapeutically Restores Oocyte Yield, Prevents Oocyte Chromosome Misalignments and Spindle Aberrations, and Increases Fertility in Midlife Female SAMP8 Mice.

Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called "egg infertility." A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women.

Proliferation of rat granulosa cells during the periovulatory interval.

Granulosa cell proliferation during luteinization and terminal differentiation has historically been assumed to decline rapidly after an ovulatory stimulus. In contrast, terminal differentiation in other cell types has recently been associated with a transient increase in proliferation, suggesting that this may occur in the ovarian follicle. The goal of the current study was to test the hypothesis that an ovulatory stimulus to rats results in additional granulosa cell proliferation before cell cycle arrest. Immature rats were given a single injection of pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) to initiate periovulatory events. The proportion of granulosa cells in S phase did not change until 12 h after hCG, although the majority of the post-hCG proliferation was localized to cumulus granulosa cells for up to 10 h after hCG. The expression of cyclin D2 mRNA did not decline until 12 h after hCG, although both cyclin-dependent kinase (Cdk)4 and Cdk6 mRNA increased at 6 h. Protein levels of cyclin D2 and Cdk4 did not change as a result of hCG, whereas cyclin E increased 6 h after hCG. Kinase activity of Cdk2 dropped markedly by 4 h after hCG, but a slight increase in activity was evident 6-8 h after hCG. These data suggest that cumulus granulosa cells continue to proliferate for up to 10 h after an ovulatory stimulus, possibly via cyclin E/Cdk2. It is concluded that proliferation is maintained in granulosa cells in the proximity of the oocyte during luteinization of the rat follicle.

The peroxisome proliferator-activated receptor gamma ligand troglitazone induces apoptosis and p53 in rat granulosa cells.

PPARgamma is expressed in both the rodent and human ovary, but the endogenous activation state of PPARgamma in the ovary and its normal role in ovarian function remain unclear. Here, we investigated mRNA and protein expression as well as DNA-binding activity of PPARgamma during follicle growth and luteinization in the immature, gonadotropin-primed rat model. Gel shift analysis demonstrated binding of ovarian PPAR to a consensus peroxisome proliferator response element (PPRE) that was supershifted with an antibody specific for PPARgamma, but not with antibodies specific for PPARalpha or beta/delta. PPARgamma expression and DNA-binding activity was highest 0-12 h post-PMSG, but declined during later stages of follicle growth (24-36 h post-PMSG). Administration of hCG induced a decline in PPARgamma mRNA, protein, and DNA-binding activity beginning at 4 h. Treatment of preovulatory granulosa cells with the PPARgamma ligand troglitazone (1-10 microM) in vitro decreased cell viability, increased sub-G1 apoptosis, and reduced DNA synthesis. Troglitazone induced p53 protein expression and decreased bcl-2 mRNA, suggesting possible mechanisms for troglitazone-induced apoptosis. These data indicate that PPARgamma is in the ovary is capable of binding DNA in the absence of pharmacological activation and provide evidence for a possible physiologic role for this receptor in regulating granulosa cell survival.

Induction of proteinases in the human preovulatory follicle of the menstrual cycle by human chorionic gonadotropin.

OBJECTIVE: To explore the temporal expression in granulosa and theca cells of key members of the MMP and ADAMTS families across the periovulatory period in women to gain insight into their possible roles during ovulation and early luteinization. DESIGN: Experimental prospective clinical study and laboratory-based investigation. SETTING: University medical center and private IVF center. ANIMAL AND PATIENT(S): Thirty-eight premenopausal women undergoing surgery for tubal ligation and six premenopausal women undergoing assisted reproductive techniques. INTERVENTION(S): Administration of hCG and harvesting of follicles by laparoscopy and collection of granulosa-lutein cells at oocyte retrieval. MAIN OUTCOME MEASURE(S): Expression of mRNA for matrix metalloproteinase (MMPs) and the A disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS) in human granulosa cells and theca cells collected across the periovulatory period of the menstrual cycle and in cultured granulosa-lutein cells after hCG. Localization of MMPs and ADAMTSs by immunohistochemistry. RESULT(S): Expression of MMP1 and MMP19 mRNA increased in both granulosa and theca cells after hCG administration. ADAMTS1 and ADAMTS9 mRNA increased in granulosa cells after hCG treatment, however, thecal cell expression for ADAMTS1 was unchanged, while ADAMTS9 expression was decreased. Expression of MMP8 and MMP13 mRNA was unchanged. Immunohistochemistry confirmed the localization of MMP1, MMP19, ADAMTS1, and ADAMTS9 to the granulosa and thecal cell layers. CONCLUSION(S): The collection of the dominant follicle throughout the periovulatory period has allowed the identification of proteolytic remodeling enzymes in the granulosa and theca compartments that may be critically involved in human ovulation. These proteinases may work in concert to regulate breakdown of the follicular wall and release of the oocyte.

Regulation of steroidogenesis by p53 in macaque granulosa cells and H295R human adrenocortical cells.

Ovulation and formation of a functional corpus luteum in primates involve cascades of events, including increased progesterone synthesis and changes in granulosa cell proliferation. However, critical gaps remain in our understanding of how an ovulatory gonadotropin surge initiates these processes. To more fully elucidate changes in the cell cycle during luteal formation, the actions of the tumor suppressor p53 were examined. Rhesus macaque granulosa cells were isolated during controlled ovarian stimulation protocols before (nonluteinized) or after (luteinized) an ovulatory gonadotropin stimulus. Phosphorylated p53 protein was detected in the cytoplasm of granulosa cells before and after human chorionic gonadotropin (hCG) treatment, whereas granulosa cells from hormonally controlled rats did not express p53 before or after hCG. Treatment of nonluteinized macaque granulosa cells with hCG and the p53 inhibitor pifithrin-alpha (PFT) in vitro did not alter markers of the cell cycle, including proliferating cell nuclear antigen, p21, and human double minute (HDM)-2 expression compared with hCG alone. Levels of pregnenolone and progesterone increased 2- and 4-fold, respectively, within 6 h of hCG treatment, whereas PFT completely blocked this hCG-induced effect. Estradiol was increased transiently (>10-fold) by hCG plus PFT relative to levels after hCG alone. PFT also inhibited hCG-induced increases in steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase mRNAs. Similar results were obtained using the human adrenocortical cell line H295R, suggesting that p53 may have a general function in primate steroidogenesis. These data indicate that p53 plays a key role in luteinization of the primate ovarian follicle though the regulation of steroidogenic enzymes leading to progesterone synthesis.

Dynamics of Myc/Max/Mad expression during luteinization of primate granulosa cells in vitro: association with periovulatory proliferation.

Granulosa cell luteinization involves the attenuation of gonadotropin-induced proliferation. Although recent evidence indicates that primate granulosa cells stop dividing within 12 h of an ovulatory stimulus, early events in cell cycle arrest remain unknown. In the current study an in vitro model of primate granulosa cell luteinization is established that allows assessment of early events in terminal differentiation. A luteinizing dose of human chorionic gonadotropin (hCG) results in a secondary rise in proliferation before cell cycle arrest that is paralleled by a transient increase in the expression of c-Myc. In contrast, the c-Myc antagonists Mad1, Mad4, and Mxi1 are transiently repressed by hCG. Max, the common dimerization partner for Myc and Mad, is similarly repressed by hCG, suggesting that changes in the expression of this gene may further regulate the activity of Myc and Mad. To determine whether other cell cycle regulatory families are involved in luteinization, the expression of p53 and the wild-type p53-inducible phosphatase (wip1) was examined. Similar to Mad and Max, p53 and wip1 are transiently repressed by hCG, suggesting that the p53 and Mad pathways have either parallel or cooperative roles in luteinization. Thus, luteinization of primate granulosa cells is preceded by a burst of proliferation that is regulated by changes in the relative levels of c-Myc, Max, and Mad as well as p53.

Dominance style of Japanese macaques compared with rhesus and stumptail macaques.

In the present study, we seek to relate dominance style with group cohesion in a captive group of Japanese macaques (Macaca fuscata). Social data were gathered on approach rate, result, and direction, aggression rate and intensity, grooming rate and direction, and conciliatory tendency. Data were collected using focal animal sampling and instantaneous scan sampling. Reconciliation data were collected using ad libitum observations of aggression with ten-minute post-conflict and matched-control focal observations. Data were compared to prior studies on rhesus (M. mulatta) and stumptail macaques (M. arctoides) living in similar environments. Each species demonstrated the presence of a formalized dominance hierarchy based on the teeth-baring display. The Japanese macaque group showed a lower rate of approach with a higher proportion of negative outcomes than either of the other species. Rates of aggression and reconciliation were also lower in the study troop, suggesting a strict hierarchy while maintaining an optimal nearest-neighbor distance. Overall, this group of Japanese macaques was less sociable than other groups of the same species, perhaps due to a history of individual removals. © 1995 Wiley-Liss, Inc.

Dietary sugar in healthy female primates perturbs oocyte maturation and in vitro preimplantation embryo development.

The consumption of refined sugars continues to pose a significant health risk. However, nearly nothing is known about the effects of sugar intake by healthy women on the oocyte or embryo. Using rhesus monkeys, we show that low-dose sucrose intake over a 6-month period has an impact on the oocyte with subsequent effects on the early embryo. The ability of oocytes to resume meiosis was significantly impaired, although the differentiation of the somatic component of the ovarian follicle into progesterone-producing cells was not altered. Although the small subset of oocytes that did mature were able to be fertilized in vitro and develop into preimplantation blastocysts, there were >1100 changes in blastocyst gene expression. Because sucrose treatment ended before fertilization, the effects of sugar intake by healthy primates are concluded to be epigenetic modifications to the immature oocyte that are manifest in the preimplantation embryo.

Shortened estrous cycle length, increased FSH levels, FSH variance, oocyte spindle aberrations, and early declining fertility in aging senescence-accelerated mouse prone-8 (SAMP8) mice: concomitant characteristics of human midlife female reproductive aging.

Women experience a series of specific transitions in their reproductive function with age. Shortening of the menstrual cycle begins in the mid to late 30s and is regarded as the first sign of reproductive aging. Other early changes include elevation and increased variance of serum FSH levels, increased incidences of oocyte spindle aberrations and aneuploidy, and declining fertility. The goal of this study was to investigate whether the mouse strain senescence-accelerated mouse-prone-8 (SAMP8) is a suitable model for the study of these midlife reproductive aging characteristics. Midlife SAMP8 mice aged 6.5-7.85 months (midlife SAMP8) exhibited shortened estrous cycles compared with SAMP8 mice aged 2-3 months (young SAMP8, P = .0040). Midlife SAMP8 mice had high FSH levels compared with young SAMP8 mice, and mice with a single day of high FSH exhibited statistically elevated FSH throughout the cycle, ranging from 1.8- to 3.6-fold elevation on the days of proestrus, estrus, metestrus, and diestrus (P < .05). Midlife SAMP8 mice displayed more variance in FSH than young SAMP8 mice (P = .01). Midlife SAMP8 ovulated fewer oocytes (P = .0155). SAMP8 oocytes stained with fluorescently labeled antitubulin antibodies and scored in fluorescence microscopy exhibited increased incidence of meiotic spindle aberrations with age, from 2/126 (1.59%) in young SAMP8 to 38/139 (27.3%) in midlife SAMP8 (17.2-fold increase, P < .0001). Finally, SAMP8 exhibited declining fertility from 8.9 pups/litter in young SAMP8 to 3.5 pups/litter in midlife SAMP8 mice (P < .0001). The age at which these changes occur is younger than for most mouse strains, and their simultaneous occurrence within a single strain has not been described previously. We propose that SAMP8 mice are a model of midlife human female reproductive aging.

Follicle growth, ovulation, and luteal formation in primates and rodents: a comparative perspective.

Ovarian function has a great deal of functional overlap between species; antral follicles grow in response to FSH, ovulation involves proteolysis, and the steroidogenic pathway is largely the same. However, embedded in these similarities are important differences that reflect the evolutionary and natural history of species and may focus future research into these critical areas. This review compares ovarian function of rats and mice with primates, focusing on estradiol and follicle growth, steroidogenesis and rupture during the periovulatory interval, and the formation of a functional corpus luteum, drawing the conclusion that careful comparison of species yields more functional information about both than studying them in isolation.

Rhesus monkey cumulus cells revert to a mural granulosa cell state after an ovulatory stimulus.

Follicular somatic cells (mural granulosa cells and cumulus cells) and the oocyte communicate through paracrine interactions and through direct gap junctions between oocyte and cumulus cells. Considering that mural and cumulus cells arise through a common developmental pathway and that their differentiation is essential to reproductive success, understanding how these cells differ is a key aspect to understanding their critical functions. Changes in global gene expression before and after an ovulatory stimulus were compared between cumulus and mural granulosa cells to test the hypothesis that mural and cumulus cells are highly differentiated at the time of an ovulatory stimulus and further differentiate during the periovulatory interval. The transcriptomes of the two cell types were markedly different (>1500 genes) before an ovulatory hCG bolus but converged after ovulation to become completely overlapping. The predominant transition was for the cumulus cells to become more like mural cells after hCG. This indicates that the differentiated phenotype of the cumulus cell is not stable and irreversibly established but may rather be an ongoing physiological response to the oocyte.

Dynamics of intra-follicular glucose during luteinization of macaque ovarian follicles.

Glucose is important to the maturation of the oocyte and development of the embryo, while hyperglycemia results in profound reproductive and developmental consequences. However, the normal physiology of glucose in the ovary remains poorly understood. The goal of this study was to determine intra-follicular glucose dynamics during the periovulatory interval in non-human primates undergoing controlled ovarian stimulation protocols. Follicular fluid and mural granulosa cells were isolated before or up to 24h after an ovulatory hCG bolus, and the human granulosa-lutein cell line hGL5 was used. Intra-follicular glucose increased 3h after hCG, and remained at that level until 12h when levels decline back to pre-hCG concentrations. Pyruvate and lactate concentrations in the follicle were not strongly altered by hCG. Mural granulosa cell expression of hexokinase 1 and 2, and glucose-6-phosphate dehydrogenase mRNA decreased following hCG, while glycogen phosphorylase (liver form) increased following hCG. Glucose uptake by hGL5 cells was delayed until 24h following stimulation. In summary, intra-follicular glucose increases following an ovulatory stimulus and mural granulosa cells do not appear able to utilize it, sparing the glucose for the cumulus-oocyte complex.