Placental growth and development is crucial for successful pregnancy. The aim of this study was to characterize the activity and localization of the matrix metalloproteinase 2 (MMP-2) and MMP-9, which are capable of degrading basement membrane collagen (predominantly collagen type IV), and their endogenous tissue inhibitor of matrix metalloproteinases (TIMPs), in amniotic fluid and in the developing ovine placenta. Cell deletion by apoptosis during placental development was also examined. Zymography with gelatin as substrate indicated that MMP-2 (72 kDa gelatinase A; predominantly latent form) was present in increasing amounts in amniotic fluid from day 70 of gestation to labour (days 140-145), and MMP-9 (92 kDa gelatinase B; predominantly latent form) was detectable from day 125 to labour; there was no increase in MMP-2 or -9 in labour. A broad range of TIMPs was detected in amniotic fluid; the molecular masses corresponded to TIMP-1, -2 and -3. Immunohistochemical techniques localized MMP-2, MMP-9 and TIMP-3 in the sheep placenta, predominantly in the trophoblast layer in uninucleate, but not binucleate, cells. However, MMP-2 and -9 activated proteins in placental homogenates were low throughout pregnancy. Apoptosis was identified by morphological criteria and also by TdT-mediated dUTP nick end labelling. Apoptosis was present in discrete regions in the placenta, predominantly in trophoblast cells near the tips and the basal regions of the fetomaternal interdigitations. During pregnancy the sheep placenta becomes more complex and the area of the fetomaternal interface increases. MMP-2 and -9 are likely to be involved in breaking down basement membranes to allow cell migration during this process. It is suggested that digestion of supporting extracellular matrix may trigger apoptosis and in some way increase the branching pattern in the villi.
SC Riley, CJ Webb, R Leask, FM McCaig and DC Howe
FH Thomas, R Leask, V Srsen, SC Riley, N Spears and EE Telfer
During ovarian folliculogenesis, ascorbic acid may be involved in collagen biosynthesis, steroidogenesis and apoptosis. The aims of this study were to determine the effects of ascorbic acid on bovine follicle development in vitro. Preantral follicles were cultured for 12 days in serum-free medium containing ascorbic acid (50 microg ml(-1)). Half of the medium was replaced every 2 days, and conditioned medium was analysed for oestradiol and matrix metalloproteinase 2 (MMP-2) and MMP-9 secretion. On day 12, cell death was assessed by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In the absence of serum, there was significant (P < 0.05) follicle growth and oestradiol secretion over the 12 day culture period. Ascorbic acid had no effect on these parameters. The addition of serum from day 0 stimulated follicle growth (P < 0.05), but compromised follicle integrity. By day 12 of culture, a higher proportion of follicles remained intact in the presence of ascorbic acid in serum-free conditions (P < 0.05), and significantly (P < 0.01) less granulosa and theca cell death was observed in these follicles than in control follicles. Moreover, ascorbic acid significantly (P < 0.05) increased production of MMP-9, an enzyme involved in basement membrane remodelling. In conclusion, this culture system was capable of supporting follicle differentiation over the 12 day culture period. Furthermore, ascorbic acid maintains bovine follicle health and basement membrane remodelling in vitro.
SC Riley, R Leask, JV Selkirk, RW Kelly, AN Brooks and DC Howe
Type 1 NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is the key enzyme for metabolism of active primary prostaglandins to inactive forms in gestational tissues. The present study examined the activity and immunolocalization of PGDH in the ovine placenta, fetal membranes and uterus over the latter half of pregnancy, and its potential regulation by oestradiol. Placenta, fetal membranes and myometrium were collected from sheep with known single insemination dates on days 70, 100 and 135 of gestation and in active labour demonstrated by electromyographic activity. In addition, chronically catheterized fetuses were infused with oestradiol (100 microgram kg(-1) per 24 h) (n = 5) or saline vehicle into the fetus from day 120 to day 125. PGDH activity measured in placental extracts remained constant from day 70 to day 135 of gestation, and then significantly (P < 0.05) increased by 300% in active labour. Immunoreactive PGDH was localized in the placentome at all stages and was present predominantly in the fetal component of the placentome in uninucleate, but not in binucleate, trophoblast cells. Similarly, in the fetal membranes PGDH immuno-reactivity was present in the uninucleate trophoblast but not in the binucleate cells of the chorion. PGDH immunostaining was also present in the endometrial luminal epithelium, in the smooth muscle of the myometrium, and the glandular epithelium of the cervix. Infusion of oestradiol into the fetal circulation from day 120 to day 125 of gestation had no effect on placental PGDH activity. Immunohistochemistry was used to localize oestrogen receptor alpha in intrauterine tissues to investigate further the failure of oestradiol to increase PGDH activity. Immunoreactive oestrogen receptor alpha was not present in the fetal component of the placenta, although it was expressed in adjacent maternal-derived cells. It is concluded that (1) PGDH activity increases in late gestation; (2) PGDH is expressed in uninucleate trophoblast cells in the ovine placenta and fetal membranes, and also in the maternal endometrial epithelium and stroma, myometrium and cervix; (3) oestrogen receptor alpha is not expressed in fetal cells in the placenta or fetal membranes; and (4) the increase in PGDH activity is not regulated by oestradiol administered to the fetus.
ED Watson, M Heald, A Tsigos, R Leask, M Steele, NP Groome and SC Riley
Ten mares were studied from February (winter anoestrus) to their second ovulation in the breeding season to investigate the relationship between resumption of ovarian cyclicity in the spring and circulating concentrations of FSH, inhibin A and inhibin isoforms containing pro- and -alphaC immunoreactivity. An additional four mares were studied during one oestrous cycle. Growth and regression of ovarian follicles were monitored by transrectal ultrasonography. The frequency of blood sampling varied from three times a week to once a day, depending on the follicular activity present. Concentrations of FSH, oestradiol, inhibin A and pro- and -alphaC isoforms were low during deep winter anoestrus when minimal follicular activity was present in the ovaries. During spring transition, an increase in FSH concentration preceded the emergence of each follicular wave. Concentrations of inhibins were significantly higher (P < 0.05) during growth of anovulatory follicles in spring transition than during winter anoestrus. Plasma concentrations of oestradiol and inhibin A were significantly higher (P < 0.001, P < 0.05, respectively) during the growth of preovulatory follicles than during the growth of transitional anovulatory follicles, but concentrations of inhibin pro-alphaC isoforms did not differ between the two types of follicle. During the oestrous cycle, there was a significant inverse relationship (P < 0.001) between concentrations of FSH and the inhibins. Plasma inhibin pro-alphaC isoforms, but not inhibin A, reached a peak on the day of ovulation. The results strongly indicate that FSH regulates growth of spring anovulatory and preovulatory follicles. Inhibins are likely to contribute to negative feedback on the release of FSH from the pituitary gland both during the transitional period and the breeding season in mares.
SC Riley, AH Gibson, R Leask, DJ Mauchline, HG Pedersen and ED Watson
Extensive tissue remodelling is required in equine ovaries for follicle growth and development and also migration of the follicle to the ovulatory fossa, where ovulation occurs. The mechanisms for these processes are largely unexplored. Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) are important for control of breakdown of extracellular matrix during tissue remodelling. The aims of this study were to determine the pattern and sites of secretion of the gelatinases MMP-2 and -9 and TIMPs into follicular fluid during follicle development in mare ovaries. The predominant gelatinase detected in follicular fluid was MMP-2, which was present in similar amounts throughout follicular development, as demonstrated by zymography. MMP-9 was also present in follicular fluid and secretion increased significantly (P < 0.05) with development of follicles from < 10 mm to 11-20 mm in diameter. Follicular fluid also contained TIMP-1, TIMP-2, unglycosylated and glycosylated TIMP-3, and TIMP-4, as shown by reverse zymography. The abundance of TIMPs remained largely unchanged during follicle development. MMP-2 and -9 were localized by immunohistochemistry to stromal cells and granulosa and theca cells. TIMP-1, -2, -3 and -4 were present in granulosa and theca cells of the follicle and in stromal cells and also associated with extracellular matrix of the ovarian stromal tissue. The MMPs and TIMPs are likely to be involved in the regulation of the breakdown of extracellular matrix during tissue remodelling for follicle development and migration to the ovulation fossa in mares.