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T. C. Wood and D. E. Wildt

Immature cumulus–oocyte complexes (COCs) were recovered from freshly excised domestic cat ovaries and graded at a magnification of × 40 for the condition of the cumulus oophorus of the oocyte cytoplasm. Grade I and II COCs were those with a uniformly dark cytoplasm and a readily identifiable, eccentrically located germinal vesicle. Grade I COCs had five or more cumulus oophorus cell layers, whereas grade II complements had less than five cell layers. Grade III and IV COCs were those undergoing progressive stages of oocyte cytoplasmic deterioration indicated by transparency or mosaic fragmentation and partial-to-complete loss of cumulus oophorus cells. In Expt 1, 699 oocytes were cultured for maturation and fertilization in vitro. More (P < 0.05) oocytes from grade I COCs matured (59.3%) and fertilized (29.7%) than from all other grades. Maturation and fertilization success did not differ (P > 0.05) for grade II (32.4, 11.6%, respectively) and grade III (21.9, 5.1%) oocytes, but these values were superior (P < 0.05) to those of grade IV (5.1, 1.4%). In Expt 2, 1040 COCs were graded, cultured for maturation and then inseminated. Of grade I oocytes, 24.4% developed into blastocysts compared with only 5.3% of grade II oocytes (P < 0.05). In general, oocytes from grade III and IV COCs were incapable of cleaving or growing in vitro. Of the 1739 COCs collected for both experiments, 12.3% met grade I criteria, the only category that provided consistent maturation, fertilization and development to blastocyst stage in vitro. In summary, a highly heterogeneous population of cumulus–oocyte complexes can be separated in the cat on the basis of grossly apparent morphological characteristics that, in turn, reflect functional differences in the ability of oocytes to mature, fertilize and develop in vitro.

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Dori C Woods and A L Johnson

While there is accumulating evidence that mitogen-activated protein kinase/Erk and protein kinase C (PKC) signaling inhibits premature differentiation of granulosa cells in hen prehierarchal follicles, it has only recently been established that these signaling pathways play an important facilitory role in promoting steroidogenesis in differentiated granulosa cells from preovulatory follicles. The present studies were conducted with differentiated granulosa cells to establish the ability of LH to initiate PKC activity, and the subsequent requirement for PKC activity in promoting the ErbB/Erk signaling cascade that ultimately facilitates LH-induced progesterone production. Incubation of differentiated granulosa cells with LH increases PKC activity within 15 min, and latently promotes Erk phosphorylation (P-Erk) by 180 min. Inhibition of PKC activity with GF109203X attenuates LH- and 8-bromo-cAMP (8-br-cAMP)-induced P-Erk, but not P-Erk promoted by an epidermal growth factor (EGF) family ligand (e.g., transforming growth factor α). Importantly, inhibition of PKC activity also blocks the LH-induced increase in the autocrine expression of mRNA encoding the EGF family ligands, such as EGF, amphiregulin, and betacellulin. Furthermore, inhibition of EGF ligand shedding at the level of the cell membrane using the matrix metalloprotease activity inhibitor, GM6001, prevents both LH- and 8-br-cAMP-induced P-Erk and progesterone production. These findings provide evidence for a facilitory role of PKC and ErbB/Erk signaling in LH-induced progesterone production, place the requirement for PKC activation upstream of ErbB/Erk activity, and demonstrate for the first time in a non-mammalian vertebrate the requirement for PKC activity in LH-induced expression of EGF family ligands in granulosa cells.

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J. C. WOOD and V. L. BARLEY

Summary.

The activities of β-glucuronidase and acid cathepsin D remain at a low level while decidual tissue is forming in the rat uterus but increase markedly during the period of decidual regression. By contrast, acid and alkaline phosphatase rise during deciduomal formation but fall during regression. The importance of lysosomal enzymes in the breakdown of decidual tissue is discussed in relation to these results.

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P. Carthew, M. Wood and C. Kirby

Summary. Mouse embryos were cultured in vitro in medium with serum containing interferon which had been induced in vivo by intravenous administration of polyinosine-polycytidylic acid. Two-cell and blastocyst-stage embryos were incubated for 72 and 24 h respectively before embryo transfer, or fixation to determine cell number. Further, blastocysts were outgrown on coverslips in embryo culture medium with fetal calf serum and interferon. Expression of an intermediate filament protein (M r 55 000) in blastocyst outgrowths was examined with a monoclonal antibody. Embryos appeared morphologically normal and after treatment the mean cell number did not differ from that of controls. Implantation was unaffected by any of the treatments, but culture before transfer in medium containing mouse serum reduced the number of normal fetuses recovered on Day 14 of gestation compared to those cultured in medium without serum. Exposure to interferon did not modify the expression of filaments in the outgrown blastocyst. It is therefore unlikely that interferon induced by viral infection during pregnancy is responsible for preimplantation embryonic loss.

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J. C. WOOD and D. R. S. KIRBY

Summary.

The presence of a thread in a limited region of the rat uterus causes a gain in weight throughout the length of the uterine horn which bears it. In addition, such a thread lessens, but does not inhibit completely, the decidual cell response which can be induced by uterine trauma. This inhibition increases progressively with the length of time that the device has been present. A mechanism is suggested to explain this effect.

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C. S. Lee, F. B. P. Wooding and M. R. Brandon

Summary. Ovine placental lactogen and the SBU-3 antigen (derived from a trophoblast membrane preparation), two proteins of widely different structure, function and destination, were shown by ultrastructural immunogold techniques to localize in identical structures in the sheep placentome throughout most of pregnancy. Both were restricted to the ultrastructurally similar membrane-bounded granules in the chorionic fetal binucleate cell and the syncytium at the fetomaternal interface. The Golgi body from which the granules form was also doubly labelled but only in the binucleate cell, never the syncytium. This provides further evidence that the binucleate cells migrate and fuse to form the syncytium. The two proteins were homogeneously distributed in the granules and would be released together by exocytosis. Only the lactogen reaches the fetal and maternal circulations so the SBU-3 may have some more local function.

In early pregnancy the SBU-3 antigen is found by itself in the granules, indicating that the association with the lactogenic hormone is not obligatory. Neither antigen was found consistently in the otherwise ultrastructurally similar interplacentomal binucleate cell granules, corroborating the presence of two functional populations of binucleate cells.

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Dori C Woods, Jeffrey S Schorey and A L Johnson

The recent identification of toll-like receptor (TLR) signaling within ovarian granulosa cells has broad implications for ovarian physiology. Functions of TLRs within granulosa cells of the laying hen are of particular interest due to the method of transovarian transmission of Salmonella enteritidis, which results in egg contamination. This study utilized hen granulosa cells to evaluate the expression and function of Gallus TLR-signaling at distinct stages of follicular maturity. Data presented herein demonstrate the presence of TLR2, TLR4, and TLR15 mRNAs in undifferentiated granulosa cells from prehierarchal follicles and differentiated granulosa cells from preovulatory follicles, together with mRNAs encoding adaptor proteins and signaling components required for TLR signaling gene. Treatment with lipopolysaccharide (LPS) or LH, in vitro, led to the differential regulation of TLRs based on the stage of follicle maturation, with the largest (F1) follicle granulosa cells having the most rapid response. Furthermore, treatment with LPS resulted in attenuation of agonist-induced progesterone synthesis in undifferentiated, but not differentiated, granulosa cells. Additionally, undifferentiated granulosa cells were significantly more sensitive to LPS-induced apoptosis than differentiated granulosa cells from the F1 follicle. Together, these data provide evidence for a complete and functional TLR signaling pathway in hen granulosa cells, with effects on steroidogenesis and cell viability dependent upon stage of maturation. These differences may reflect the susceptibility of granulosa cells at early stages of maturation to undergo apoptosis in response to select pathogenic stimuli, thus attenuating transovarian transmission, whereas granulosa cells from preovulatory follicles are comparably resistant to LPS-mediated apoptosis.

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Rosemary C. Bonney, Deborah J. Wood and Devra G. Kleimant

Summary. Urinary excretion of oestrogens and androgens by a pair of giant pandas was monitored by radioimmunoassay during behavioural oestrus through two successive breeding seasons. The excretion of oestrogens by the female was at a maximum during the proceptive period and lower during the period of receptivity. In the first breeding season studied, elevated androgen excretion in the male coincided with peak receptivity in the female.

The study indicates that accurate timing of natural mating or artificial insemination could be achieved by monitoring oestrogen excretion in the female.

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M. Tuffrey, F. Alexander, C. Woods and D. Taylor-Robinson

Summary. Groups of mice from genetically defined inbred strains were infected genitally with a pathogenic human strain of Chlamydia trachomatis and their subsequent fertility was compared. The CBA, C3H (H-2°) and C3H/He-mg (H-2 k) mice were less fertile than control mice, at least up to 6 months after infection. In contrast, fertility was not impaired in BALB/c mice or in congenic BALB/K mice, which had the H-2 k haplotype. Reduced fertility was paralleled by the extent of histological oviductal inflammation in mice of each strain. No salpingitis was seen 21 days after infection in the BALB strains, but lesions were apparent in CBA and C3H strains up to about 70 days after inoculation and these sometimes developed into hydrosalpinges. These results indicate that susceptibility to chlamydial salpingitis and subsequent infertility is under genetic control. This control was not simply associated with the major H-2 gene complex, as mouse strains of the same haplotype (H-2 k) differed in susceptibility. The fertility of BALB/c (H-2 d) and BALB/K (H-2 k) strains was no different from that of controls, and congenic C3H mice of differing H-2 haplotypes (H-2 k and H-2°) showed reduced fertility. Although all the infected F1 (BALB/K × C3H/He-mg) mice produced litters at the same rate as untreated controls, the litters were considerably smaller. This was due to the occurrence of unilateral pregnancies in the mice inoculated under the ovarian bursae and possibly also to early fetal death in mice inoculated directly in the uterus. These findings emphasize the importance of early diagnosis and treatment of infection of the lower genital tract of women.

Keywords: Chlamydia; infertility; genetics; mouse

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OJ Ginther, BG Woods, C Meira, MA Beg and DR Bergfelt

Follicle growth and circulating hormone concentrations were compared between an interovulatory interval and the first 60 days of the anovulatory season in pony mares. Daily observations were made from November of three groups: (i) ablation of follicles of >/=6 mm in diameter at day 10 after an ovulation that initiated an interovulatory interval, as determined retrospectively (ovulatory group, n=8), (ii) ablation at day 10 after the last ovulation of the year (anovulatory-10 group, n=6); and (iii) ablation at day 60 after the last ovulation of the year (anovulatory-60 group, n=6). Follicular waves were defined as major (dominant follicle) and minor (no dominant follicle). The percentage of mares with major waves after ablation for the ovulatory, anovulatory-10 and anovulatory-60 groups was 100, 33 and 0%, respectively, and the percentage with minor waves was 0, 67 and 100%, respectively. Minor waves were also detected in 83% of anovulatory mares between day 20 and day 60. Growth of the largest follicle was similar for major waves and minor waves but only until the beginning of deviation in the major waves. FSH surges after ablation were similar for all groups and for surges detected during days 20-60. Concentrations of LH were greater in association with major waves than with minor waves. Both diameter of the largest follicle and LH concentrations for minor waves were greater after ablation at day 10 after the last ovulation of the year than after ablation at day 60. The results of this study indicate that major follicular waves developed in some mares early in the anovulatory season and that minor waves developed throughout the first 2 months. Despite similarities in the wave-stimulating FSH surge, differences in follicle growth occurred and were attributable, on a temporal basis, to differences in LH concentrations. A minor wave developed into a major wave when the largest follicle reached a diameter characteristic of the beginning of deviation in the presence of an adequate LH stimulus for continued growth of a dominant follicle.