P. J. Hansen

Involvement of apoptosis in disruption of developmental competence of bovine oocytes by heat shock during maturation.

Abstract Various pathological stimuli such as radiation, environmental toxicants, oxidative stress, and heat shock can initiate apoptosis in mammalian oocytes. Experiments were performed to examine whether apoptosis mediated by group II caspases is the cause for disruption of oocyte function by heat shock applied during maturation in cattle. Bovine cumulus-oocyte complexes (COCs) were cultured at 38.5, 40, or 41°C for the first 12 h of maturation. Incubation during the last 10 h of maturation, fertilization, and embryonic development were at 38.5°C and 5% (v/v) CO2 for all treatments. In the first experiment, exposure of COCs to thermal stress during the first 12 h of maturation reduced cleavage rate and the number of oocytes developing to the blastocyst stage. In the second experiment, a higher percentage of TUNEL-positive oocytes was noted at the end of maturation for oocytes matured at 40 and 41°C than for those at 38.5°C. In addition, the distribution of oocytes classified as having high (>25 intensity units), medium (15–25 intensity units), and low (<15 intensity units) caspase activity was affected by treatment, with a greater proportion of heat-shocked oocytes having medium or high activity. In the third experiment, COCs were placed in maturation medium with vehicle (0.5% [v/v] DMSO) or 200 nM z-DEVD-fmk, an inhibitor of group II caspases. The COCs were matured at 38.5 or 41°C, fertilized and cultured for 8 days. The inhibitor blocked the effect of heat shock on cleavage rate and the percentage of oocytes and cleaved embryos developing to the blastocyst stage. In conclusion, heat shock during oocyte maturation can promote an apoptotic response mediated by group II caspases, which, in turn, leads to disruption of the oocytes capacity to support early embryonic development following fertilization.

Identification of Possible Mediators of Embryonic Mortality Caused by Mastitis: Actions of Lipopolysaccharide, Prostaglandin F2α, and the Nitric Oxide Generator, Sodium Nitroprusside Dihydrate, on Oocyte Maturation and Embryonic Development in Cattle

Problem: Mastitis and immunization against constituents of organisms causing mastitis can reduce fertility of cattle and sheep, respectively. For the current experiments, it was hypothesized that these effects are mediated via actions of lipopolysaccharide (LPS), prostaglandin F2α (PGF2), and nitric oxide on oocyte maturation and embryonic development.

Actions of Tumor Necrosis Factor-α on Oocyte Maturation and Embryonic Development in Cattle1

Problem:  Infertility can accompany mastitis in cattle. Involvement of tumor necrosis factor‐α (TNF‐α) in this phenomenon is suggested by observations that circulating concentrations of TNF‐α are elevated after intramammary infection or infusion of endotoxin. It was hypothesized that (1) TNF‐α acts on the oocyte during maturation to decrease the percent of oocytes that cleave and develop following fertilization; (2) exposure of embryos to TNF‐α after fertilization reduces development to the blastocyst stage; and (3) TNF‐α increases the proportion of blastomeres that undergo apoptosis in a stage‐of‐development dependent manner.

Inhibition of lymphocyte proliferation by ovine trophoblast protein-1 and a high molecular weight glycoprotein produced by the peri-implantation sheep conceptus

ABSTRACT: Sheep conceptuses were flushed from uteri on day 16 of pregnancy and cultured in vitro. Three peaks of immunosuppressive activity (i.e., ability to inhibit [3H]thymidine incorporation into mitogen‐stimulated lymphocytes) were localized in conceptus‐conditioned culture medium: one corresponding to a high‐molecular‐weight glycoprotein (HMWG; Mr = 800–900 kDa), one corresponding to ovine trophoblast protein‐1 (oTP‐1), the antiluteolytic, interferon‐like molecule of the sheep conceptus, and a third fraction containing a previously undescribed molecule with a molecular weight between 10 and 14 kDa. Both HMWG and oTP‐1 inhibited lymphocyte proliferation in a dose‐dependent manner. Delaying the addition of HMWG or oTP‐1 until 24 h after the addition of phytohemagglutinin (PHA) reduced the degree of suppression relative to cultures where HMWG and oTP‐1 were added at the time of PHA addition. Nonetheless, the molecules were still capable of inhibiting proliferation. Addition of human recombinant IL‐2 (0–65 U/ml) to PHA‐stimulated lymphocyte cultures could not reverse HMWG‐ or oTP‐1‐induced suppression of [3H]thymidine uptake by PHA‐stimulated cells. Furthermore, treatment of lymphocytes with HMWG or oTP‐1 suppressed IL‐2‐induced proliferation. Therefore, HMWG and oTP‐1 affect both early and later events in the in vitro proliferative response of mitogen‐stimulated lymphocytes, including responsiveness to IL‐2.

Effect of injection of β-carotene or vitamin E and selenium on fertility of lactating dairy cows

Abstract Experiments tested whether supplemental antioxidants improved fertility. To test effects of β-carotene, cows in a hot environment were injected with prostaglandin F 2α (PGF 2α ) and were given 3 injections, im, of 800 mg β-carotene or saline at Days -6 and -3 before the anticipated date of insemination and at insemination (n=37−41 inseminated cows/group). There was no effect of β-carotene on the proportion of cows detected in estrus following PGF 2α , timing of estrus after PGF 2α injection or pregnancy rate in inseminated cows. In a second trial, cows in a temperate climate received intramuscular injections of vitamin E (500 mg) and selenium (50 mg) at 30 d post partum (n=97) or were untreated controls (n=89). Treatment did not affect interval from calving to first insemination or the proportion of cows pregnant at first service, but it increased the pregnancy rate at second service (69.8 vs 52.1%; P=0.07) and reduced services per conception (1.7 vs 2.0; P

Fibroblast growth factor 10 enhances bovine oocyte maturation and developmental competence in vitro

The ability of oocytes to resume meiosis, become fertilized, and generate viable pregnancies is controlled during folliculogenesis by several endocrine and paracrine factors. The aim of this work is to determine whether fibroblast growth factor 10 (FGF10) is an oocyte competent factor. Transcripts for each of the four FGF receptor types (FGFR) were present in cumulus and oocytes after their extraction from the follicles. FGFR1 transcripts predominated in cumulus cells whereas FGFR2 was most abundant in oocytes. Exposing the cumulus-oocyte complexes to FGF10 during in vitro maturation did not affect cleavage rates, but increases (P<0.05) in the percentage of embryos at the 8-16-cell stage on day 3 and at the blastocyst stage on day 7, which were evident in FGF10-supplemented oocytes. The progression of oocytes through meiosis and cumulus expansion was increased (P<0.05) by FGF10. The importance of the endogenous sources of FGFs was examined by adding anti-FGF10 IgG during oocyte maturation. Blocking endogenous FGF10 activity decreased (P<0.05) the percentage of oocytes developing into blastocysts and limited (P<0.05) cumulus expansion. Expression profiles of putative cumulus and oocyte competency markers were examined for their involvement in FGF10-mediated responses. FGF10 influenced the expression of CTSB and SPRY2 in cumulus cells and BMP15 in oocytes. In summary, this work provides new insight into the importance of FGFRs and locally derived FGF10 during oocyte maturation in cattle. Its subsequent impact on in vitro embryo development implicates it as a noteworthy oocyte competent factor.

Inhibition of lymphocyte proliferation by bovine trophoblast protein-1 (Type I trophoblast interferon) and bovine interferon-αI1

Bovine trophoblast protein-1 (bTP-1) is a Type I interferon secreted by the bovine trophoblast from about Day 15 of pregnancy. It is not known whether bTP-1 has functional properties in common with other interferons. The aim of the present study was to determine whether bTP-1 inhibits proliferation of lymphocytes induced by mitogens, mixed lymphocyte cultures (MLC) and interleukin-2 (IL-2) and, if so, whether this activity is similar to that of a related interferon, bovine interferon-alpha I1 (bIFN-alpha I1). Stimulation of lymphocyte proliferation caused by phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) was inhibited by bTP-1 and bIFN-alpha I1 without any reduction in cell viability. Maximum or near-maximum inhibition (less than 50%) was achieved at concentrations of 0.5-5.0 nM of bTP-1 and bIFN-alpha I1. Cells stimulated with PWM were less inhibited than cells stimulated with PHA and Con A. Both bTP-1 and bIFN-alpha I1 inhibited MLC to a greater degree than lectin-stimulated cells (maximum inhibition was 78% or greater). Also, bTP-1 and bIFN-alpha I1 slightly inhibited incorporation of [3H]thymidine ([3H]TdR) induced by the combination of phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), and calcium ionophore A23187. Finally, bTP-1 and bIFN-alpha I1 had bimodal effects on incorporation of [3H]TdR by IL-2-induced lymphocytes. Incorporation of [3H]TdR was increased at 0.005 nM and 0.05 nM concentrations while higher concentrations caused a slight decrease in [3H]TdR incorporation. Results confirm that bTP-1 inhibits lymphocyte proliferation in a manner similar to that caused by the leukocyte-derived interferon, bIFN-alpha I1. Incomplete inhibition of mitogen-induced proliferation and differences in degree of inhibition between various stimulators suggest that bTP-1 and bIFN-alpha I1 preferentially inhibit certain lymphocyte subpopulations. Local inhibition of lymphocyte proliferation caused by bTP-1 may help protect the allogeneic conceptus from immune responses to fetal antigens or regulate the release of cytokines from endometrial lymphocytes.

Presence of an intracellular endometrial inhibitor of prostaglandin synthesis duing early pregnancy in the cow

Previous studies have detected reduced endometrial secretion of prostaglandins during pregnancy in cattle. The present experiment tested the hypothesis that reduced secretion of prostaglandins is caused by induction of an intracellular endometrial inhibitor of prostaglandin synthesis. The microsomal fraction of parturient bovine cotyledons was utilized as a source of enzymes for prostaglandin synthesis. Endometrial tissues collected at Day 17 of the estrous cycle (n = 12) and pregnancy (n = 12) were homogenized and subjected to differential centrifugation for preparation of microsomes and a high-speed (100,000 x g) cytosolic supernatant. Endometrial intracellular preparations were then examined for the ability to modulate prostaglandin synthesis by cotyledonary microsomes from parturient cows. Endometrial intracellular preparations from cyclic cows decreased (P less than 0.05) PGF synthesis by cotyledonary microsomes to a slight extent (supernatant, 21% reduction; microsomes, 11% reduction), while preparations from pregnant cows markedly decreased (P less than 0.01) PGF synthesis (supernatant, 63% reduction; microsomes, 28% reduction; supernatants vs microsomes, P less than 0.01). Regardless of the amount of arachidonic acid available as substrate (25-400 micrograms) endometrial supernatant from pregnant cows (pooled sample) caused a 50% inhibition (IC50) of prostaglandin synthesis at a tissue equivalent of 270 +/- 9.1 mg. The mechanism of inhibition by endometrial high-speed supernatant from pregnant cows appears to be non-competitive with respect to arachidonic acid. The inhibitor(s) may be proteinaceous (70-75 kDa and 25-35 kDa) and can be precipitated by 20% saturated ammonium sulfate. In conclusion, early pregnancy in cattle appears to be associated with increased amounts of an intracellular endometrial inhibitor of prostaglandin synthesis.

Effects of stage of the estrous cycle and steroid treatment on uterine immunoglobulin content and polymorphonuclear leukocytes in cattle

Studies were performed to determine 1) cyclic changes in uterine immune function that could be responsible for the increased susceptibility of the diestrous uterus toinfection and 2) whether these changes are mediated by progesterone or estradiol-17#. The number and functional activity (cytochrome c reduction) of polymorphonuclear leukocytes (PMNL) collected from the uterus after oyster glycogen infusion was not statistically affected by stage of the cycle or steroid treatment. While not significant, the number of PMNL tended to be higher at diestrus than at estrus. The number of PMNL also tended to be higher for ovariectomized cows treated with progesterone than with estradiol-17/3 or vehicle. For cows given oyster glycogen, uterine IgG was not sitmificantly affected by treatment and uterine IgA was undetectable. For ovariectomized cows not given oyster glycogen, IgA was detectable in uterine flushings and was higher after 30 d of progesterone treatment than after 12 d of progesterone or vehicle. Additionally, the ability of uterine fluid from ovariectomized cows to inhibit mitogen-induced lymphocyte proliferation was greater (P < 0.01) for uterine fluid collected from cows treated with progesterone for 12 and 30 d than for cows treated with vehicle. It is concluded that alterations in migration of PMNL are unlikely to be the cause of decreased resistance of the uterus to infection during diestrus. Lowered resistance might, in part, be due to the secretion of uterine factors that inhibit lymphocyte function.

Localization of granulocyte-macrophage colony-stimulating factor in the bovine reproductive tract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) can increase embryo development to the blastocyst stage in cattle. The objective of the present study was to determine whether GM-CSF is present in the reproductive tract. Using Western blotting, immunoreactive GM-CSF was detected in uterine flushings from cows at days 0, 7, and 14 of the estrous cycle and from cows at days 14-17 of pregnancy. Also, GM-CSF was localized immunohistochemically to endometrium and oviduct. Patterns of immunohistochemical localization and intensity of reaction product were similar for all days of the estrous cycle. While present in several cell types, immunoreactive product in the endometrium was greatest in epithelium (especially luminal epithelium). Immunoreactive GM-CSF was also localized to epithelium in ampullary and isthmic regions of the oviduct, with intensity greater in ampulla. Staining was observed for both ciliated and non-ciliated cells. In conclusion, the bovine oviduct and endometrium contain immunoreactive GM-CSF and this molecule is present in uterine secretions. Thus, this cytokine is a potentially important intracellular regulator of endometrial, oviductal and embryonic function during early pregnancy in the cow.