Relationship between maternal plasma progesterone concentration and interferon-tau synthesis by the conceptus in cattle

ELSEVIER

RELATIONSHIP BETWEEN MATERNAL PLASMA PROGESTERONE CONCENTRATION AND INTERFERON-TAU SYNTHESIS BY THE CONCEPTUS IN CATTLE T.L. Kerbler,’ M.M. Buhr,’ L.T. Jordan,’ K.E. Leslie* and J.S. Walton’ Departments of ‘Animal and Poultry Science, ‘Veterinary Microbiology and Immunology, and 3Population Medicine, University of Guelph Guelph, Ontario, Canada N 1G 2 W 1 Received for publication: Accepted:

September

11,

1995

SepLember

i9,

i996

ABSTRACT The objective of this study was to determine the relationship between maternal progesterone concentration and conceptus synthesis of interferon-r as an index of conceptus viability at the time of maternal recognition of pregnancy. Heifers of mixed beef breeds were randomly assigned to receive 1 of 2 treatments: 1) intramuscular injection of 1500 IU hCG on Day 5 after artificial insemination (AI; n=12) or 2) intramuscular injection of saline on Day 5 after AI (n=17). Ovaries were scanned daily by transrectal real-time ultrasonography. Progesterone concentrations were determined from daily blood samples collected l?om the jugular vein. Heifers were slaughtered on Day 18 afler AI and conceptus tissues were collected. These were incubated individually at 37°C in RPMI medium, and supernatant collected after 24 h. Conceptus secretory products in the supematant were analyzed for interferon concentration by antiviral assay using vesicular stomatitis virus. Transrectal ultrasonography showed all heifers that received hCG had at least 1 extra corpus luteum (CL) in addition to the spontaneous CL formed from the previous ovulation (10 with 2 CL, 2 with 3 CL). A significant increase in plasma progesterone concentration was detected in pregnant heifers treated with hCG (n=9) vs pregnant control heifers (n=l 1; P < 0.001). There was a tendency for an increase (P=O.O59) in synthesis of interferon-r by conceptuses horn hCG-treated heifers compared to control heifers. Maternal plasma progesterone concentrations were correlated with interferon-r production by the conceptuses (r = 0.593 P < 0.006), suggesting that higher maternal progesterone may provide a more suitable environment for the developing conceptus. 0

1997 by Elsevw Scxnce Inc

Key words: human chorionic gonadotrophin, progesterone, pregnancy, interferon-z Acknowledgments This study was supported by the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture, Food, and Rural Affairs. The authors are indebted to Michelle Goodwin for technical assistance and to W. Szkotnicki for statistical support. Charlie Watson and the staff at the Elora Beef Research Station are thanked for providing animal care and assistance, and the Meat Technology Laboratory of the Department of Animal and Poultry Science is gratefully acknowledged for slaughtering the animals and providing tissues. Theriogenology 47:703-714. 0 1997 by Elsevier Science

1997 Inc.

0093~691x/97/$17.00 PII s0093-691x(97)00028-9

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INTRODUCTION High rates of embryo mortality continue to be a major cause of reproductive failure in cattle (25). It has already been established that progesterone is required to maintain a suitable uterine environment for the healthy development of the conceptus (1829). A number of approaches increasing progesterone levels have been studied. The use of progesterone-releasing intravaginal devices (PRID; 42,45) has proven successll in increasing progesterone levels and pregnancy rate. The PRIDs, however, are expensive, disturb vaginal microflora and drainage (4), and are Increasing subject to societal concerns about the use of exogenous steroid hormones. progesterone levels endogenously by the induction of accessory CL has proven to be a less expensive and potentially more attractive alternative. An injection of 1000 to 1500 IU hCG 5 d post breed& causes the ovulation of one or more large follicles leading to supplementary CL and a consistent increase in progesterone concentrations over a sustained period (19,38,39,45). Trophoblast protein-l, also known as interferon-r (IFN-z) (41), is a complex of glycoproteins secreted in large quantities by the conceptus between Days 17 to 22 of gestation (2,13,14) that act in an antiluteolytic manner to either inhibit or alter the pattern of endometrial release of prostaglandin F-2a to remove its luteolytic effects (15,20,2 1). In addition, IFN-t also possesses potent antiviral and anti-proliferative activities (36,37,40) which assist maintenance of pregnancy. Since it has been shown that conceptuses at different stages of development produce different amounts of IFN-z (14,22), the production of IFN-t, expressed as anti-viral activity, can, therefore, be used as a measure of conceptus development and viability. The primary objective of this research was to determine if increasing maternal plasma progesterone concentrations via a single injection of hCG would provide a more suitable environment for the development of the conceptus as demonstrated by an increase in IFN-r synthesis at the time of maternal recognition of pregnancy. MATERIALS AND METHODS Animals Twenty-nine crossbred beef heii housed at the Elora Beef Research Station were treated with 2 intramuscular injections of cloprostenol(5OOpg; Estrumate, Cooper’s Agropharm Inc., Ajax, ON, Canada) 11 d apart, and were observed 3 d later for signs of estrus. The heifers were artiiicially inseminated with previously frozen semen from a single ejaculate of a Shorthorn bull (Strathmore’s Extra Edition, United Breeders Inc., Guelph, ON, Canada) 72 h a&r the last cloprostenol injection. Progesterone and Ovarian Ultrasonography Beginning at the second cloprostenol injection, blood samples were collected daily by jugular venipuncture into hep arinized tubes, and cooled on ice. Plasma was separated within 3 h and stored at -20°C for progesterone analysis. Plasma progesterone concentrations were

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determined using the solid phase radioimmunoassay (RIA) previously described by Robinson et al. (42). The intra- and inter-assay coefficients of variation were 8.8 and 11So?, respectively. Daily transrectal ultrasonography commenced 2 d following insemination. Ultrasound examinations of the ovaries were performed using a linear array real-time ultrasound device (Aloka Echo Camera Model SSD-210-DX, Aloka Co., Tokyo, Japan) equipped with a 5 MHZ rectal probe (Aloka Probe Model LIST-58 13-5). The ovaries were scanned in several planes to identify all follicles greater than 1 mm in diameter, and to provide an image of the CL with its greatest cross sectional area. A hard copy was made of all appropriate images with a video printer (Mitsubishi Video Copy Processor, Tokyo, Japan), and growth of follicles and CLs were charted over time. Conceptus Isolation and Culture Eighteen days after insemination the heifers were slaughtered by captive bolt pistol and exsanguination, and the reproductive organs immediately removed. The ovaries were removed, and CLs dissected and diameters measured. The vagina was severed at the external OSof the cervix and both uterine horns were simultaneously flushed with 35 ml PBS introduced into the oviducts. The flushings were collected into petri dishes. Conceptuses were transferred individually into petri dishes containing 15 ml RPMI medium 1640, with L-glutamine and without L-leucine (Gibco BRL, Life Technologies Inc., Burlington, ON, Canada) supplemented with 2% penicillin and streptomycin (G&co) and were incubated (Model 3860, Forma Scientific Inc.. Marietta, OH, USA) for 24 hat 37°C in 5% CO, / 95% air (35). After culture, the medium was collected, divided into 5-ml aliquots and stored at -20°C until analyzed for anti-viral activity using an interferon anti-viral assay by plaque reduction adapted f?om Loewen and Derbyshire (27). Inoculum of culture supematant (0.4 ml) was placed on 1-d-old monolayers of Madin-Darby bovine kidney celIs (MDBK)(VMI, OVC, Guelph, ON, Canada) and incubated in 5% CO, / 95% air, at 37°C for 18 h. After this IFN pretreatment, the plates were washed and 40 to 60 plaque-forming units of vesicular stomatitis virus (vsv) were added to each well. The plates were incubated under the previous conditions for 1 h, then the vsv was removed and the plates washed with PBS (0.1 M phosphate, 9% sodium chloride, 0.1% sodium azide). One milhhtre of 0.9% tragacanth overlay (VMI, OVC, Guelph, ON, Canada) was added to each plate and plates were incubated for 48 h. A 10% formalin solution in PBS was used to fix each well, and crystal violet stain was used for plaque observation. Antiviral titre was determined by counting plaques and recording the dilution at which there was at least a 50% reduction in the number of plaques relative to the virus control wells. Antiviral titres were related to a laboratory porcine leucocyte IFN standard and a human recombinant IFN-a,, (Roferon, Hofhnan-La Roche, Mississauga ON, Canada). Each culture supematant was tested in duplicate at 2 or more dilutions, with an intra-assay coefficient of variation of 15%. Statistical Analysis To determine the et&t of hCG treatment on progesterone concentrations, the progesterone profiles were analysed by calculating the arca under the curve using the trapezoid rule. The areas

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were then subjected to the General Linear Model procedure of the Statistical Analysis System (SAS; 43) to test for treatment efkcts. DiEerences in CL diameter were analyzed using Student’s t-tests. The IFN-r data were normalized using a natural log transformation and analyzed using the General Linear Model procedure of SAS to determine differences between treatments. The regression procedure of SAS was used to determine the regression of IFN-r synthesis by conceptuses with area under the maternal progesterone profiles. RESULTS Ovarian Changes After Treatment Injection of hCG on Day 5 induced ovulation of the large dominant follicle which grew after insemination, as evidenced by the diippearance of the follicle, followed by the appearance of an additional CL observed by ultrasound on Days 10 to 11, and present at slaughter on Day 18 in all treated cows. Ten of the 12 heiters treated with hCG had 2 CL, with the remaining 2 heifers treated with hCG having 3 CL (Table 1). Measurements using real-time ultrasonographic images detected a difference (PcO.05) in CL diameter Gram Day 10 until slaughter between the induced CL and the spontaneous CL in pregnant hCG-treated heifers (Figure 1). The diameters of the spontaneous CL in hCG-treated heifers and control heifers were similar from Day 10 until slaughter. Measurements taken on Day 18 confirmed differences between the induced CL (2.4 * 0.15 cm) and the spontaneous CL (2.7 f 0.13 cm) in pregnant hCG-treated heifers. The dieters of the spontaneous CL of the hCG-treated (2.7 f 0.13 cm) and the control heifers (2.4 f 0.08 cm) were Shlih.

Table 1. Pregnancy rates and luteal status of heifers on Day 18 a&r treatment with 1500 IU hCG or saline on Day 5 after insemination Observations

Pregnant Non Pregnant Mean no. of CL No. with 1 CL No. with 2 CL No. contralateral No. ipsilateral No. with 3 CL

Treatment hCG

Saline

9 (75%) 3 (25%)

11 (64.7%) 6 (35.3%)

2.2 + 0.11 0 10 4 6 2

# Pregnant 7 (70%) 3 (75%) 4 (67%) 2 (100%)

1 17 0

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Figure 1. Mean CL diameter (cm) for pregnant heifers treated with 1500 IU hCG (n=9) or saline (n=l 1) on Day 5. *Induced CL (hCG[I]) di&rs t?om spontaneous CL (hCG[S]).

Plasma Progesterone Plasma progesterone concentrations were simihtr from Day 1 to Day 5 (Figure 2), and there was a significant increase in heifers treated with hCG compared with that of controls (P
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Figure 3. l[nte&ron Tau synthesized after 24 hour culture of Day 18 bovine conceptuses from heikrs treated with hCG (1500 IU; n=9) or saline (sl 1) on Day 5 after insemination.

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Area Under Progesterone Curve Day 5 To 18 (arbitrary units) Figure 4. Positive correlation between maternal plasma progesterone concentration and interferon Tau synthesis by Day 18 conceptuses (n=20) after 24-hour culture.

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The development of the accessory CL was similar to that of the spontaneous CL (10). This induced luteal structure appears to develop normally, since no dh%mnces have been detected between the CL of hCG-treated cows and control cows in luteal weights, DNA, RNA or protein concentrations (10). Price and Webb (38) found the hCG-induced CL to be smaller than the spontaneous CL, attributing the size difference to the difference in the ages of the CL. In contrast, Rajamahendran and Sianangama (39) found treatment with hCG leads to significant increases in total diameter of the CL, with the induced CL becoming difficult to distinguish from the spontaneous CL. The induced CL in this study , however, grew to a smaller diameter than the spontaneous CL confirming the work of Price and Webb (38). The reduced size of the induced CL could be due to a smaller follicle being induced to ovulate by hCG. With normal ovulation, the preovulatory follicle According to ultrasound has 2 or 3 additional days for growth as it progresses to ovulation. observations (not shown), the dominant follicle was still increasing in size at the time of hCG injection. This was followed by a sudden disappearance of the follicle, followed 2 to 3 days later by the appearance of a rapidly growing but smaller CL at the same location. A significant increase in plasma progesterone concentrations occurred between Days 8 and 20 post insemination after hCG treatment on Day 5. The effect of hCG was not apparent until around Day 8 post estrus, by which time accessory CL are formed and produce supplementary progesterone (10,45). In this experiment the accessory CL were not evident by ultrasonography until Day 10 to 11, but increased progesterone concentrations were detectable by Day 8. The latter may have been due to an effect of hCG on the spontaneous CL. Since, ovulation occurs 20 to 25 h after the LH surge (6), it does not seem likely that the increase in progesterone concentration detected by Day 8 is the result of the luteal structure formed by hCG administration on Day 5. It has been reported that increases in plasma progesterone (8,11,26) after hCG administration were due to a direct effect of hCG on hypertrophy (8) and or to increased blood flow to the CL (34). Farin et al. (8) found that treatment with hCG can also alter the population of large and small luteal cells, since small luteal cells may differentiate into large luteal cells. As luteinization of both CL continues, progesterone production by the small luteal cells is responsive to LH (17,23); any diirentiation of small to large luteal cells plus the additional large cell population in the induced CL leads to larger amounts of progesterone being secreted by LH-independent mechanisms (48) and the increased plasma progesterone concentrations observed. Interferon-r synthesis tended to be greater in conceptuses from heifers treated with hCG (P
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of progesterone advanced uterine secretions so that a Day 6 uterine environment was suitable for development of a Day 10 embryo (26,44). Exogenous progesterone also induces similar advancements in uterine environment and conceptus development in cattle (12). In the current study, interferon-z measurement was on a conceptus basis and it is uncertain whether the effect of progesterone was due to increased interferon-r synthesis or advanced conceptus development. Conceptuses were cultured immediately to avoid compromising their viability. There were, however, no obvious differences between treatments in the size or development of the conceptuses which would suggest the effect is on synthetic ability. Nephew et al. (32) recently also found conceptuses fiorn ewes treated with hCG secreted more IFN-r than conceptuses from control ewes. A precedent for this relationship between progesterone concentration and synthesis of interferon-r by the conceptus has also been provided by observations that pregnant heifers have higher progesterone concentrations than non-pregnant heifers during early pregnancy (5,7,16,28). This may indicate the importance of progesterone to conceptus survival or vice versa. The amount of interferon-r synthesized by the conceptus is influenced by the development status of the embryo, and may be used as an objective indicator of embryo quality since it provides a direct measure of trophoblast biosynthetic activity (1,22). It is also possible that hCG may have a direct influence on endometrial function and, therefore, indirectly on conceptus development. The recent observation of the presence of endometrial receptors for hCG/LH in the bovine endometrium early in the estrous cycle (9) indicates that such a possibility cannot be excluded. Nevertheless, the strengthened relationship resulting from the association of progesterone with interferon-z without concern for hCG treatment may indicate that the indirect effect through progesterone is more important than any hCG effects per se. In this and other studies (39,45), the effect of hCG treatment on pregnancy rates has generahy been equivocal. There is generally a small increase in pregnancy rate after hCG, but this is not signilicant perhaps because of inadequate sample sizes, The data from this study are encouraging in demonstrating that maternal plasma progesterone concentrations are strongly associated with the abii of the conceptus to synthesize a secretory product known to be important for the recognition of pregnancy. Further studies will be necessary to determine if these observations are consistent with the use of hCG treatment to increase pregnancy rates. In conclusion, these results show that the large early luteal-phase follicle is capable of responding to hCG to produce an accessory CL, which allows for an endogenous increase in plasma progesterone concentration in the cow. This increase in progesterone concentration would appear to be beneficial to the development of the conceptus as seen by the strong correlation between progesterone concentration and interferon-s synthesis by the conceptus. REFERENCES 1.

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