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B. K. Tsang, M. Li and J. A. Carnegie

Summary. The secretion of progesterone and 20α-hydroxypregn-4-en-3-one (20α-dihydroprogesterone) by granulosa cells from 30-day-old rats pretreated with PMSG (4 i.u.; i.p.) was significantly increased in a time- and concentration-dependent manner by FSH or cytochalasin B. Whereas FSH markedly stimulated progestagen secretion during 3 h of incubation, a significant enhancement of the steroidogenic response was not noted until 12 h of exposure to the inhibitor in vitro. Although cytochalasin B also enhanced the submaximal stimulation of progestagen production by FSH (15 ng/ml), it was ineffective in the presence of maximal stimulatory concentration of the gonadotrophin (150 ng/nl). With increasing concentrations of cytochalasin B, the ability of FSH to further stimulate progestagen secretion was progressively reduced. Granulosa cells cultured in medium alone contained a prominent cytoplasmic array of microfilaments which was markedly reduced by FSH or cytochalasin B. FSH and, to a greater extent, cytochalasin B elicited concentration-dependent reductions in the mean area occupied by the cells on the culture surface, the contour index (a size-independent representation of cell profile irregularity) and cell perimeter, indicating that the cells underwent less spreading and were more spherical and regular in outline in the presence of either agent. The FSH-induced reductions in the three shape-related parameters were augmented by cytochalasin B although the influence of the FSH on the mean area and perimeter was progressively reduced in the presence of higher concentrations of cytochalasin B. These findings are consistent with the concept that microfilaments influence cell shape and steroidogenesis in granulosa cells in vitro and that FSH alters microfilament distribution and shape of cultured granulosa cells in eliciting its steroidogenic influence.

Keywords: microfilaments; steroidogenesis; FSH; granulosa cells

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Deidre M Mattiske, Li Han and Jeffrey R Mann

RNA interference (RNAi) has diverse functions across cellular processes, including a role in the development of the mammalian oocyte. Mouse primary oocytes deficient in the key RNAi enzyme DICER1 exhibit pronounced defects in chromosome congression and spindle formation during meiotic maturation. The cause of this meiotic maturation failure is unknown. In this study, observations of chromosomes and spindle microtubules during prometaphase in DICER1-deficient oocytes indicate that chromosome congression and spindle formation are overtly normal. Spindle breakdown and chromosome displacement occur after the metaphase plate has formed, during the metaphase to anaphase transition. We hypothesised that this defect could be attributed to either RNAi-mediated regulation of nuclear factors, such as the regulation of centromere chromatin assembly, or the regulation of mRNA expression within the cytoplasm. By transplanting germinal vesicles between DICER1-deficient and wild-type primary oocytes, we show that, unexpectedly, the meiotic failure is not caused by a deficiency derived from the germinal vesicle component. Instead, we reveal that the ooplasm of primary oocytes contains DICER1-dependent factors that are crucial for chromosome segregation and meiotic maturation.

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B. K. Tsang, D. F. Mattice, M. Li and E. K. Asem

Summary. The gonadotrophic regulation of progesterone production by rat granulosa cells was examined in a chemically-defined medium containing FSH, dibutyryl cyclic AMP ((Bu)2cAMP) and the calcium ionophore, A23187. FSH and A23187 alone significantly enhanced the production of pregnenolone, progesterone and its metabolite, 20α-hydroxypregn-4-en-3-one (20α-OH-P) from endogenous substrate(s). Stimulation of progesterone production by A23187 was accompanied by an increase in 3β-hydroxysteroid dehydrogenase (3β-HSD) but not 20α-hydroxysteroid dehydrogenase (20α-HSD) activity, as attested by enhancement of the metabolism of exogenous pregnenolone to progesterone but not of progesterone to 20α-OH-P. In contrast, although (Bu)2cAMP increased pregnenolone and progesterone production and the metabolism of exogenous progesterone to 20α-OH-P, it failed to stimulate the conversion of exogenous pregnenolone to progesterone. The increase in progesterone production and in the conversion of exogenous pregnenolone to progesterone by FSH and A23187 was concentration- and time-dependent. Whereas maximal stimulation of de-novo progesterone synthesis by FSH was evident by 6 h (earliest time examined), a significant increase in the conversion of exogenous pregnenolone to progesterone in the presence of FSH or the ionophore was not noted until 12 h of incubation. Although a small but significant increase in progesterone production was also noted as early as 6 h of incubation in the presence of the calcium ionophore, this was markedly smaller than that elicited by FSH.

We conclude that the calcium ionophore A23187 and (Bu)2cAMP have similar as well as distinct effects on progesterone production in rat granulosa cells in vitro. We suggest that, while cAMP may be involved in the more rapid control by gonadotrophin of the production of the steroid via increased synthesis of pregnenolone and/or its metabolism to 20α-OH-P, calcium may be important in the synthesis and metabolism of pregnenolone for the maintenance of steroidogenic capacity of the granulosa cells.

Keywords: calcium; cAMP; 3β-hydroxysteroid dehydrogenase; 20α-hydroxysteroid dehydrogenase; steroidogenesis; granulosa cell; gonadotrophin action; rat

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Jaime Hughes, Wing Yee Kwong, Dongfang Li, Andrew M Salter, Richard G Lea and Kevin D Sinclair

We previously reported increased follicular fluid progesterone (P4) concentrations in ewes fed an n-3 compared to an n-6 polyunsaturated fatty acid (PUFA)-enriched diet, but detected no differential effect of n-3 and n-6 PUFA-enriched high-density lipoproteins (HDL) on granulosa cell (GC) steroidogenesis in vitro. Moreover, net n-6 PUFA-enriched HDL reduced early embryo development, but in the absence of a net uptake of FA. Consequently, we hypothesised that a) effects of n-3 PUFA on ovarian steroidogenesis are mediated by theca rather than GCs and b) during embryo culture lipids are acquired solely from the albumin fraction of serum, so that albumin-delivered n-3 and n-6 PUFA exert a greater differential effect on embryo development than either low-density lipoprotein (LDL)- or HDL-delivered PUFA. Data confirmed that n-3 PUFA increases P4 production solely in theca cells and that this is associated with an increase in STAR transcript expression. Furthermore, LDL- and HDL-delivered n-3 PUFA are equally efficacious in this regard during the first 96 h of culture, but thereafter only HDL-delivered n-3 PUFA induces this effect in partially luteinised theca cells. We also demonstrate that albumin is the sole serum fraction that leads to a net uptake of FA during embryo culture. PUFA-enriched serum and albumin increased the yield of morphologically poorer quality blastocysts with increased transcript expression for the antioxidant enzyme SOD1. Important differential effects of n-3 and n-6 PUFA on ovarian steroidogenesis acting solely on theca cells are identified, but differential effects of PUFA on embryo development are less apparent.

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M. K. Vaughan, S. Oaknin, B. Cozzi, K. Li and R. J. Reiter

Summary. Daily afternoon injections of 25 μg melatonin for 12 weeks had no effect on testicular weights of male rats kept in long photoperiod (14L:10D); similarly, exposure of rats to short photoperiod (2L:22D) had no effect on gonadal weight. However, rats maintained in a long or short photoperiod and implanted every 2 weeks with a 15 mm Silastic pellet containing testosterone showed a significant reduction in testicular weight; this effect was more pronounced in rats exposed to a short photoperiod. Melatonin injections in testosterone-treated rats in a long photoperiod exacerbated the inhibitory effects of testosterone alone. Subcutaneous 2-weekly implants of a beeswax pellet containing 1 mg melatonin reversed the effects of the melatonin injections on relative testicular weights but not those due to short photoperiod exposure.

Testosterone implants significantly reduced pituitary LH values in long and short photoperiod-exposed animals, more particularly in those exposed to short photoperiod. Melatonin injections alone or in combination with melatonin pellets did not further exaggerate the depression in pituitary LH due to testosterone alone in long photoperiod-exposed animals; similarly melatonin pellets did not reverse the depression in pituitary LH observed. No significant differences in plasma prolactin concentrations or in thyroxine concentrations or free thyroxine index were observed after any combination of treatments.

We therefore suggest that the effects observed with short photoperiod may be due to melatonin.

Keywords: melatonin, testosterone pellets, photoperiod, LH, prolactin, T4, T3

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T. C. Li, M. A. Warren, P. Dockery and I. D. Cooke

Department of Obstetrics and Gynaecology, University of Sheffield, Jessop Hospital for Women, Sheffield S3 7RE, UK, and Department of Biomedicai Science, University of Sheffield, Sheffield S10 2TN, UK

Introduction

Grosser (1910) stated that the most important physiological function of the endometrial cycle is the preparation for the reception of a fertilized ovum (implantation), while menstruation is only a secondary process, a degeneration of the 'mucus membrane' which has not been able to fulfil its purpose. From the earlier work of Hertig et al. (1956), it is thought that implantation starts ∼6 days after ovulation. Croxatto et al. (1978) found that, in the natural cycle, the human embryo usually arrives in the endometrial cavity 96 h or more after the luteinizing hormone (LH) surge. In most in-vitro fertilization programmes, fertilized embryos are transferred to the endometrial cavity a day or two earlier, which is still compatible with successful implantation.

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C. J. Huang, Y. Li, M. H. Stromer and L. L. Anderson

Summary. Insulin-like growth factor I (IGF-I) is involved in paracrine/autocrine regulation of gonadal steroidogenesis and peptide hormone biosynthesis. This study was designed to determine whether IGF-I alone, or an interaction of IGF-I, is involved in augmenting the actions of luteinizing hormone (LH) and prolactin in controlling relaxin and progesterone secretion from ageing corpora lutea of hysterectomized gilts at days 110, 113 and 116 after oestrus. Luteal tissue slices were incubated for 8 h with IGF-I (0, 50, 300 ng ml−1), LH (0, 100, 1000 ng ml−1), and prolactin (0, 100, 1000 ng ml−1) alone or in combination. Progesterone and relaxin concentrations were determined by radioimmunoassay of spent medium and of homogenates from luteal tissue slices before and after incubation. Porcine luteal tissue from day 110 had a net output of 25 ng progesterone and 26 ng relaxin in the control and of 65 ng progesterone and 2125 ng relaxin in the combined IGF-I, LH and prolactin treatment mg−1 of luteal tissue, respectively. IGF-I, LH and prolactin alone or in combination significantly increased (P<0·01) progesterone production by luteal tissue from day 110, but they were partially effective at day 113 and ineffective at day 116. By contrast, the same hormone treatments increased relaxin production by luteal tissue from days 110 and 113. Even at day 116, prolactin alone or with LH or IGF-I continued to stimulate relaxin production. In conclusion, IGF-I augments the ability of prolactin and LH to increase relaxin production by ageing corpora lutea; however, a decrease in progesterone secretion and an increase in relaxin secretion at day 113 indicate that different mechanisms control progesterone and relaxin secretion in pigs.

Keywords: IGF-I; gonadotrophins; relaxin; progesterone; ageing luteal cells; pig

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L. S. Leshin, S. M. P. Raj, C. K. Smith, S. C. M. Kwok, R. R. Kraeling and W. I. Li

Pig seminal proteins PSP-I and PSP-II are major protein components of boars' ejaculate and are present as heterodimers (PSP-dimer) in seminal plasma. These proteins were examined for their ability to modulate pig lymphocyte activity in vitro in mitogen-induced lymphocyte proliferation assays and in one-way mixed lymphocyte reactions. Pig lymphocytes were cultured with phytohaemagglutinin, concanavalin A, or pokeweed mitogen (PWM) in the presence or absence of pig seminal proteins and the amount of cellular [3H]thymidine was used as an indication of proliferation. In the absence of mitogens, none of the three pig seminal proteins affected lymphocyte proliferation suggesting that these proteins are not antigenic or mitogenic. PSP-dimer enhanced lymphocyte proliferation induced by PWM (156–227%, P < 0.05) in a concentration-dependent manner, but had no effect on phytohaemagglutinin- or concanavalin A-induced proliferation. PSP-I enhanced (127–185%, P < 0.05) phytohaemagglutinin-induced proliferation. PSP-II augmented (130–240%, P < 0.05) lymphocyte proliferation induced by concanavalin A and PWM. Lymphocytes from gilts were significantly more responsive to concanavalin A- and PWM-induced lymphocyte proliferation in the presence of PSP-I compared with boars (concanavalin A: gilts 131%, boars 91%; PWM: gilts 188%, boars 134%; P < 0.05). In the mixed lymphocyte reaction, pretreating stimulating cells with increasing concentrations of PSP-I or PSP-II elicited a 400% concentration-dependent increase (P <0.01) in lymphocyte proliferation. The abundance of pig seminal proteins in boar seminal plasma, their ability to enhance lymphocyte proliferation, and their previously reported ability to bind to lymphocytes suggest that these proteins are immunostimulatory and supports the hypothesis that they modulate uterine immune activity to ensure reproductive success.

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S. M. Laird, C. F. Dalton, M. A. Okon, R. A. D. Bunning, R. Marshall and T. C. Li

The presence of metalloproteinase activity in endometrial flushings obtained from premenopausal women, during the proliferative and secretory phases of the menstrual cycle, control post-menopausal women and women with post-menopausal bleeding (PMB) with or without adenocarcinoma was analysed by zymography. In addition, quantitative measurements of matrix metalloproteinase 2 (MMP-2), MMP-3, MMP-9 and tissue inhibitor of metalloproteinase 1 (TIMP-1) in the flushings were obtained by ELISA. The zymography results showed eight bands of activity, with molecular weights ranging from 51 to 208 kDa in the flushings from pre-menopausal women and post-menopausal women, particularly those with adenocarcinoma. Both zymography and ELISA showed that MMP-2 and MMP-9 were the major metalloproteinases found in the flushings and only low concentrations of MMP-3 were found. Concentrations of MMP-2 in pre-menopausal women were higher in flushings obtained during the secretory phase of the menstrual cycle than those obtained in the proliferative phase (P < 0.05), suggesting that it may play a role in embryo implantation. Concentrations of MMP-2 (P < 0.001), MMP-9 (P <0.05) and TIMP-1 (P < 0.001) in the flushings from post-menopausal control women were lower than those from pre-menopausal women. Concentrations of MMP-2 (P < 0.05) and TIMP-1 (P < 0.05) were higher in flushings from women with PMB without carcinoma compared with post-menopausal controls and concentrations of MMP-9 (P < 0.01) and TIMP-1 (P < 0.05) in flushings from women with adenocarcinoma were higher than in post-menopausal controls. Among subjects with PMB, concentrations of MMP-9 in women with adenocarcinoma were higher than those without carcinoma (P < 0.05). Our results show that concentrations of MMP-2, MMP-9 and TIMP-1, but not MMP-3, are associated with endometrial activity and, therefore, may have a role in the breakdown of endometrial tissue. In addition, the increased concentrations of MMP-9 in flushings of women with adenocarcinoma indicate that this particular proteinase is associated with the presence of endometrial neoplastic cells.

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Y-F Liu, M-Y Li, Y-P Yan, W Wei, B Li, H-Y Pan, Z-M Yang and X-H Liang

Maintenance of a suitable uterine milieu is important for embryo development and subsequent implantation during early pregnancy. High estrogen level in proestrous and estrous stages is essential for uterine anti-bacterial activity during preimplantation period. Lipocalin-2 is an essential molecule which prevents bacterial infection by sequestering iron. In this study, the highest expression of lipocalin-2 is observed in the endometrial epithelium on day 1 of normal pregnancy and pseudopregnancy, which exhibit a similar hormone scenario. By injecting the agonists for estrogen receptor α and estrogen receptor β in ovariectomized mice, we found estrogen receptor α is the dominant member for estrogen regulation on lipocalin-2 expression. Estrogen treatment in estrogen receptor α-knockout mice further confirmed the role of estrogen receptor α. Using published data from whole-genome estrogen receptor α binding site assay, significant estrogen receptor α recruitment peaks are found at the downstream of lipocalin-2 gene after estrogen treatment. Furthermore, to study the anti-bacterial activity of lipocalin-2 in uterus, Escherichia coli is injected to mimic bacterial infection. Our results showed an obvious induction of lipocalin-2 in Escherichia coli-treated group. Taken together, this study indicates estrogen regulation of lipocalin-2 in uterine epithelium is mediated by estrogen receptor α, and lipocalin-2 may have anti-bacterial activity during early pregnancy.