Meiotic maturation in mammalian oocytes is a complex process which involves extensive rearrangement of microtubules, actin filaments and chromosomes. Since cytoskeletal elements are sensitive to disruption by heat shock, a series of experiments were performed to determine whether physiologically relevant heat shock disrupts the progression of the oocyte through meiosis, fertilization and zygote formation. Cumulus–oocyte complexes were cultured at 38.5, 40.0 or 41.0 °C for the first 12 h of maturation. Incubation during the last 10 h of maturation and 18 h after fertilization was at 38.5 °C and in 5% (v/v) CO2 for both treatments. Examination of the cytoskeleton and the chromosome organization in matured oocytes revealed that oocytes matured at 38.5°C were mostly at metaphase II (MII) stage, while the majority of heat-shocked oocytes were blocked at the first metaphase (MI), first anaphase or first telophase stages. A subset of heat-shocked oocytes possessed misshapen MI spindles with disorganized microtubules and unaligned chromosomes. A higher percentage of TUNEL-positive oocytes was noted for oocytes matured at 41.0 °C. Addition of 50 nmol/l sphingosine 1-phosphate to maturation medium blocked the effect of heat shock on progression through meiosis and apoptosis and increased the proportion of oocytes matured at 41.0 °C that were at MII. Following insemination, a high percentage of heat-shocked oocytes were unfertilized, while the majority of the control zygotes were fertilized and had two visible pronuclei. In conclusion, heat shock disrupts nuclear maturation and induces apoptosis. These alterations are likely to be involved in the mechanism underlying heat-shock-induced disruption of oocyte capacity for fertilization and subsequent development.
Lilian J Oliveira and P J Hansen
The presence of conceptus alloantigens necessitates changes in maternal immune function. Here, we used the cow to evaluate whether species with epitheliochorial placentation have changes in specific leukocyte populations during pregnancy similar to those reported in species with hemotropic placentae. At days 33–34 of pregnancy, there was no effect of pregnancy status on the number of cells positive for CD8, CD4, γδT cell receptor, or the monocyte marker CD68 in the peripheral blood mononuclear cell (PBMC) population. There was, however, an increase in the proportion of CD4+ cells that were positive for CD25. There was no effect of status on the proportion of PBMCs that were CD8+ when comparing preparturient cows to nonpregnant cows. However, preparturient cows had an increased percentage of PBMCs that were γδT cells and CD4+CD25+ and a tendency for a lower percentage that were CD68+ cells. Using immunolocalization with anti-CD68, it was found that pregnant cows had increased numbers of CD68+ cells in the endometrial stroma as early as days 54–100 of gestation; this increase persisted through the last time examined (day 240 of gestation). Cells positive for CD68 were also positive for another macrophage/monocyte marker, CD14. In conclusion, pregnancy in the cow is associated with changes in peripheral and endometrial leukocyte numbers, which are similar to patterns observed in other species.
J. R. Malayer, P. J. Hansen, and W. C. Buhi
Summary. Endometrial explant cultures were prepared from 16 Brahman × Angus cows killed on Days 0, 2, 5 or 8 after oestrus. Cultures proceeded for 24 h at 39°C (homeothermic) or 43°C (heat shock) in a modified Eagle's minimal essential medium supplemented with 50 μCi l-[4,5-3H]leucine. Analysis by two-dimensional polyacrylamide gel electrophoresis of de-novo synthesized proteins secreted into the medium indicated that the major types of secreted polypeptides did not change over Days 0–8. Nevertheless, overall endometrial secretion of protein (incorporation of [3H]leucine into non-dialysable radioactivity in culture supernatants) was greatest at Day 0 and declined thereafter. Incorporation of [3H]leucine into TCA-precipitable material in tissue homogenates was also greatest at Day 0. For tissue cultured at 39°C, several individual polypeptides were secreted at greater rates by endometrium from the horn of the uterus ipsilateral to the corpus luteum, with side differences tending to be greatest at Day 0 or Day 2. Overall, secretion of de-novo synthesized protein by endometrium was significantly elevated by heat shock at Day 0, but not affected thereafter. Nonetheless, heat shock reduced secretion of several individual proteins and exhibited interactions with day of the oestrous cycle and with side of the uterus. Secretion of 7 polypeptides was reduced by heat shock in tissue from the ipsilateral horn of the uterus but not in endometrium from the contralateral horn. We suggest that endometrial protein secretion changes quantitatively during the early oestrous cycle. In addition, there is a local influence of the ovary bearing the corpus luteum on endometrial function that may be disrupted by heat shock.
Keywords: cow; uterus; endometrium; oestrus; heat shock; protein secretion
P. J. Hansen, F. W. Bazer, and R. M. Roberts
Summary. In one experiment, ovariectomized gilts were treated with corn oil (vehicle), progesterone, oestradiol-17β or both steroids. While oestradiol treatment did not stimulate enzyme activity in uterine flushings relative to vehicle-treated animals, gilts treated with progesterone had elevated amounts of all enzymes measured. Progesterone was less effective when co-administered with oestradiol-17β. Enzymes were not equally stimulated by progesterone. For example, there was a 909-fold increase in acid phosphatase activity in uterine flushings and a 304-fold increase in β-N-acetylglucosaminidase, but only a 10-fold increase in β-glucosidase. Endometrial explants from gilts synthesized and secreted radiolabelled β-N-acetylglucosaminidase, suggesting that at least some lysosomal enzymes enter the uterus through secretory processes. In other experiments, changes in β-N-acetylglucosaminidase in uterine fluids of mares and ewes treated with hormonal regimens similar to those given to the gilts were evaluated. Treatment with the combination of progesterone and oestrogen stimulated accumulation of the enzyme relative to that in vehicle-treated animals. The biochemical properties of porcine β-N-acetylglucosaminidase were examined in detail. Properties of the uterine enzyme were similar to reported values for lysosomal hexosaminidase. These included molecular weight (82 000–89 000), pH optimum (pH 4·4), presence of two isomers (isoelectric points of 5·5 and 8·0) and ability to hydrolyse substrates for glucosaminidase and galactosaminidase. We conclude that steroids induce the accumulation of lysosomal enzymes in the uterine lumen. The degree of stimulation differed between enzymes, suggesting that those enzymes stimulated to the greatest extent may play an important role in pregnancy.
C. Plante, W. W. Thatcher, and P. J. Hansen
Summary. An experiment was conducted to (i) determine whether administration of recombinant bovine interferon-αI 1 (rBoIFN-α) attenuates oxytocin-induced release of prostaglandin F-2α and (ii) confirm previous observations that rBoIFN-α causes acute changes in body temperature and circulating concentrations of progesterone. Cows were treated twice a day from Day 14 to Day 17 after oestrus with a control regimen (bovine serum albumin (BSA), i.m. + BSA intrauterine (i.u.)), rBoIFN-α, i.u. + BSA, i.m. (rBoIFN-IU) or rBoIFN-α, i.m. + BSA, i.u. (rBoIFN-IM). On Day 17, plasma concentrations of 13,14-dihydro,15-keto-prostaglandin F-2α (PGFM) were measured after injection of oxytocin. Cows treated with rBoIFN-IU and rBoIFN-IM had longer oestrous cycles and luteal lifespans than control cows. A hyperthermic response and decline in plasma concentrations of progesterone was noticed after administration of rBoIFN-α on Day 14. On other days, the hyperthermic response was not present and the decline in progesterone was less pronounced. There was no significant effect of rBoIFN-α on circulating concentrations of oestradiol between Days 14 and 17. The release of PGFM induced by oxytocin was lower in cows treated with rBoIFN-α than in control cows. Oxytocin caused increased plasma concentrations of PGFM in four of five control cows, two of five rBoIFN-IU cows and two of five rBoIFN-IM cows. The peak PGF-2α response to oxytocin (peak value after injection minus mean concentration before injection) was 257·8 ± 60·3 pg/ml for control cows, 100·7 ± 40·8 pg/ml for rBoIFN-IU and 124·9 ± 40·4 pg/ml for rBoIFN-IM. It is concluded that rBoIFN-α can reduce oxytocin-induced PGFM release and may therefore extend the lifespan of the corpus luteum by interfering with events leading to luteolytic release of PGF from the uterus. Administration of rBoIFN-α can cause acute changes in body temperature and circulating concentrations of progesterone that become less severe after repeated exposure to rBoIFN-α.
Keywords: interferon; endometrium; prostaglandin F-2α; oestrous cycle; corpus luteum; cow
S. D. Helmer, P. J. Hansen, W. W. Thatcher, J. W. Johnson, and F. W. Bazer
Summary. Intrauterine infusion of enriched bovine trophoblast protein-1 complex (bTP-1) resulted in extension of interoestrous intervals and corpus luteum function in cyclic cattle. Conceptus proteins were obtained by culture of Day 17–18 conceptuses for 72 h. Media from the first (n = 28), second (n = 26) and third (n = 19) 24 h of conceptus incubations were utilized. A highly enriched preparation of bTP-1 was obtained by a combination of ammonium sulphate precipitation, ion-exchange chromatography, and h.p.l.c. gel filtration. Degree of purity of the final preparation was confirmed by gel electrophoresis and immunoblotting with antiserum to ovine trophoblast protein-1. Jersey cattle (3 per group) received intrauterine infusions, twice daily from Day 15·5 to 21·0, of bovine serum albumin, the entire array of bovine conceptus secretory proteins (bCSP) from the 3 days of conceptus culture, or bTP-1. Infusions were via a catheter into the uterine horn ipsilateral to the corpus luteum. Oestrous cycle length in bTP-1-treated cows 26·1 ± 1·3 days) was greater than for cows given BSA (19·5 ± 1·3 days) or bCSP (21·5 ± 1·3 days). Similarly, progesterone concentrations in serum remained elevated for a longer period of time for bTP-1-treated cows than for cows treated with BSA or bCSP. Residual variance associated with vena cava concentrations of PGF-2α at Days 19–21 after oestrus (which included the variance between 15-min periods within cows) was reduced in cows treated with bTP-1 as compared to other groups. Lack of a bCSP effect may have been due to low amounts of bTP-1 in conceptus-conditioned medium from cultures of >24 h. None the less, purified bTP-1 was effective in extending luteal function and appears to be the antiluteolytic agent of early pregnancy.
Keywords: cattle; trophoblast protein-1; oestrous cycle; intrauterine infusion; corpus luteum; prostaglandins; luteolysis
B. G. Low, P. J. Hansen, M. Drost, and K. J. Gogolin-Ewens
Summary. Immunohistochemistry was utilized to determine expression of the major histocompatibility complex (MHC) antigens on Day 8–9 hatched blastocysts and fetal membranes of mid- to late gestation cows and to examine the pattern of leucocytic infiltration into the gravid uterus. Hatched blastocysts were weakly positive for MHC class I antigens. In the mature placenta, chorioallantoic membranes in the interplacentomal area showed positive immunostaining for class I antigens on the chorionic epithelium but had no staining for class II antigens. There was an accumulation of lymphoid cells expressing class II antigens directly beneath the luminal epithelium of the endometrium. In addition, cells staining for leucocyte common antigen were present both within and beneath the luminal epithelium. Some cells positive for class II and leucocyte common antigen (CD45) were also associated with uterine glands. In the placentomes, class I antigens were expressed only on maternal caruncular septa. Fetal cotyledonary villi had no detectable immunostaining for class I and II antigens. No distinct pattern of leucocyte infiltration in the maternal caruncular tissue was observed; the caruncular septa contained some cells that were labelled for CD45 and a few class II-positive cells around blood vessels. The results indicate that the fetal placenta of the cow expresses MHC class I antigens in a regionally defined manner and there is a differential accumulation of lymphoid cells in the uterus.
Keywords: placenta; cow; major histocompatibility complex antigens; uterus; leucocyte
K. B. Finchert, F. W. Bazer, P. J. Hansen, W. W. Thatcher, and R. M. Roberts
Summary. In Exp. I, 0·5 mg oestradiol or vehicle (0·5 ml absolute ethanol + 0·5 ml 0·9% NaCl) was injected i.v. at 08:00 h on Day 14 (onset of oestrus = Day 0). Blood samples were obtained via a jugular catheter at 30 and 1 min before oestradiol and every 30 min for 10 h afterwards. Plasma was obtained and assayed for 15-keto-13,14-dihydro-PGF-2α (PGFM) by radioimmunoassay. Before oestradiol, PGFM basal values were higher (P < 0·01) in pregnant (N = 10) than nonpregnant (N = 6) ewes (193 ± 30 vs 67 ± 8 pg/ml). However, at 4–10 h after oestradiol, pregnant ewes (N = 5) had less variable (P < 0·01) PGFM values than did nonpregnant ewes (N = 5). In Exp II, conceptus secretory proteins (CSP) were obtained by pooling medium from cultures of Day-16 sheep conceptuses (N = 40). Ewes received 750 μg CSP + 750 μg plasma protein (N = 6) or 1500 μg plasma protein (N = 6) per uterine horn at 08:00 h and 18:00 h on Days 12–14. All ewes received 0·5 mg oestradiol at 08:00 h on Day 14 and blood samples were collected as in Exp. I and assayed for PGFM. On Day 15, 3 ewes in each group received 10 i.u. oxytocin and 3 received saline i.v. at 08:00 h and blood samples were taken continuously from 10 min before to 60 min after treatment. Mean PGFM response to oestradiol was suppressed (P = 0·05) in CSP- vs plasma protein-treated ewes (371 ± 129 vs 1188 ± 139 pg/ml). Oxytocin induced a greater (P < 0·01) PGFM peak response in plasma protein-treated (3972 ± 2199 pg/ml) than CSP-treated (1669 ± 287 pg/ml) ewes. Uterine production of PGF in response to oestradiol was suppressed in pregnant and CSP-treated ewes and oxytocin-induced PGF production was also suppressed in CSP-treated ewes. These results are consistent with the theory that CSP act to prevent luteolysis by altering either the amount of PGF released by the uterus or the pattern of PGF release.
S. D. Helmer, T. S. Gross, G. R. Newton, P. J. Hansen, and W. W. Thatcher
Summary. Evidence exists that conceptuses alter endometrial protein secretion and modulate prostaglandin (PG) synthesis and secretion in cattle. The present study was designed to test the effect of bovine conceptus secretory proteins (bCSP) in general and the bovine trophoblast protein-1 complex (bTP-1) in particular on endometrial function. Endometrial explants from cyclic cows (N = 4) at Day 17 after oestrus were incubated for 24 h with 0, 4·8, 24 or 120 μg BSA/ml, 1 μg bTP-1/ml or 12·7 μg bCSP/ml. Both bCSP and bTP-1 decreased (P < 0·005) release of radiolabelled macromolecules into medium and incorporation of radio-labelled precursors into tissue compared to BSA-treated tissues but not tissues treated with medium alone. Secretion of a protein of M r = 14 900 was enhanced by bCSP treatment as compared to treatment with bTP-1 (P < 0·025). Both bCSP and bTP-1 decreased PGF secretion of explants (P < 0·01) compared to BSA. Overall, PGE-2 secretion by bCSP- and bTP-1-treated tissues did not differ from that of BSA cultures, but PGE-2 secretion was greater (P < 0·025) for bTP-1 than bCSP-treated endometrium. Cotyledonary microsomes from parturient cattle were utilized as a PG-generating system for the detection of inhibitors of PG synthesis. PGF synthesis by the generating system was decreased (P < 0·05) by 9% by cytosol from BSA-treated explants, whereas cytosol from bCSP-and bTP-1-treated explants decreased (P < 0·01) PGF synthesis by 42% and 35%, respectively. In summary, both bCSP and bTP-1 inhibit PGF secretion, induce synthesis of an intracellular inhibitor of PGF synthesis, and decrease protein synthesis and secretion. The bTP-1 complex therefore alters PG dynamics by explants in a manner that would function to prevent luteolysis and support the establishment of pregnancy.
Keywords: endometrium; cattle; conceptus proteins; prostaglandins; prostaglandin synthesis inhibitor
S. D. Helmer, P. J. Hansen, R. V. Anthony, W. W. Thatcher, F. W. Bazer, and R. M. Roberts
Summary. This paper demonstrates that a group of proteins, representing a major secretory component of cattle conceptuses, is immunologically related to ovine trophoblast protein-1 (oTP-1), a principal product of culture Day 13 to 21 sheep conceptuses. Conceptuses from cows (Day 17–18) and ewes (Day 16–17) were cultured for 24 h in the presence of l-|3H]leucine. By using a rabbit antiserum to oTP-1 and Ouchterlony double-immunodiffusion analysis it was shown that material in the bovine conceptus culture medium was serologically related, but not identical, to oTP-1. A solid-phase radiobinding assay indicated that the cross-reacting bovine secretory component had an affinity for anti-oTP-1 antibody similar to that of oTP-1. Anti-oTP-1 antiserum specifically immunoprecipitated a group of 6–8 polypeptides from culture medium of cow conceptuses which, when analysed by two-dimensional gel electrophoresis, fell into two major molecular weight classes (22 000 and 24 000) with isoelectric points between 6·5 and 6·7. These immunoprecipitated polypeptides, defined as bTP-1, constituted the major secretory products of Day 16–25 cow conceptuses. They were larger and more basic than oTP-1 polypeptides (M r about 18 000; pI 5·4–5·7). Anti-oTP-1 antiserum also recognized the major translation product of Day 17 bovine conceptus mRNA, a polypeptide significantly smaller (M r ∼ 18 000) than the secreted protein. It is suggested that oTP-1 and the homologous bovine protein may play similar roles in the phenomenon of maternal recognition of pregnancy in the two species.