Delayed luteolysis and suppression of testosterone secretion after recombinant ovine interferon treatment in goats (Capra hircus). A. M. Homeida and A. I. Al- ...
Delayed luteolysis and suppression of testosterone secretion after recombinant ovine interferon treatment in goats (Capra hircus) A. M. Homeida and A. I.
Al-Afaleq
College of Veterinary Medicine and Animal Resources, King Faisal University,
PO Box 1757,
Al-Ahsa 31982, Saudi Arabia
Oxytocin at a dose of 100 iu injected
s.c. daily into goats (Capra hircus) between day 3 and of the oestrous cycle caused a significant increase in testosterone secretion and day 6 luteolysis compared with saline-treated animals. Intrauterine administration of recombinant ovine interferon tau (80, 160 or 320 \g=m\gday \m=-\1) between days 12 and 18 of the oestrous cycle, or concomitantly (80 \g=m\gday \m=-\1) with oxytocin between day 3 and day 7, delayed luteolysis and blocked the increased release of testosterone. It is suggested that recombinant ovine interferon tau can act as an antiluteolytic agent in goats.
Materials and Methods
Introduction In
goats (Homeida and Cooke, 1982) and
most other
species
(guinea-pigs, cows, ewes) in which the nonpregnant uterus has a luteolytic action (Anderson et al, 1969; Martal, 1981), the presence of the embryo prevents luteolysis. The antiluteolyic factors secreted by sheep, goat and cattle conceptuses are closely related structurally to a-interferons (IFN-a) and are known as ovine, caprine and bovine trophoblast protein-1, respectively (Gnatek et al, 1989; Plante et al, 1990; Ott et al, 1991). These embryonic interferons bind to IFNreceptors, have antiviral and antiproliferative properties and can influence the production of uterine PGF2a and proteins (Bazer et al, 1987; Roberts, 1989). Intrauterine administra¬ tion of IFN- can extend the interoestrous interval and luteal lifespan in cows (Plante et al, 1991) and sheep (Stewart et al, 1992). Significant improvement in the pregnancy rate has been observed in unilaterally ovariectomized ewes treated with IFN- (Nephew et al, 1990). An increase in embryonic survival and the promotion of maternal recog¬ nition of pregnancy in sheep (Schalue-Francis et al, 1991) are also among the many biological activities of trophoblast interferons. Another process by which luteal regression is elicited in the goat is via testosterone release (Homeida and Cooke, 1984; Homeida, 1986). Testosterone may be released in response to PGF2(I secreted by the uterus, since both hormones are synchronously released during luteal regression and are further stimulated by oxytocin and blocked by oxytocin antagonists during oxytocin-induced luteolysis (Homeida and Khalafalla, 1990). The objective of the present experiment was to determine the effects of recombinant ovine interferon on luteal function and on natural and oxytocin-induced testosterone secretion in goats.
Animals
Twenty-eight Awassi goats (2—4 years of age, and with a normal oestrous cycle of 19-20 days) were used. They were housed in individual pens under conditions of natural daylength and temperature. The oestrous cycles were synchron¬ ized with two 5 mg injections i.m. of a PGF2(1 analogue (Lutalyse: Upjohn Ltd, Crawley) given 11 days apart; when they exhibited oestrus (determined by a fertile buck), they were randomly allocated to the groups described below.
Experiment
1
In group A, four goats were injected daily with oxytocin s.c. a dose of 100 iu between day 3 and day 6 of the oestrous cycle (oestrus day 0). In group B, four goats were treated like those in group A but were given saline instead of oxytocin [0.9% (w/v) NaCl]. In group C, four goats were treated like those in group A, but in addition recombinant ovine interferon tau (roIFN- ) (a gift from Professor F. W. Bazer, Texas) was infused i.u. at a dose of 40 µg twice per day, divided equally between the two horns. The protein was mixed with 3 mg BSA and saline; it was about 90% pure and had 0.45 IO8 antiviral 1 units mg~ (Ott et al, 1991). Animals were anaesthetized i.v. at
=
with sodium oxygen
thiopentone
At surgery
(Fincher
et al, 1986), a sterile polyvinyl catheter was inserted 30 mm into the anterior lumen of each uterine horn. Catheters were secured to the flank with suture.
Experiment 2 In group D, the uteri of four goats were infused with 3 mg BSA in saline between day 12 and day 18 of the oestrous cycle. 4 goats in each group), the animals In groups E, F and G ( were treated like those in group D but were infused twice a day with 40, 80 and 160 µg roIFNT, respectively. =
Received 9 March 1994.
and maintained with fluothane and
(Homeida and Khalafalla, 1990).
Table 1. Mean ( ± sd) plasma concentration of progesterone and testosterone in (group A), saline (group B) and oxytocin and recombinant ovine interferon tau
oestrous
Progesterone (ng ml :)
cycle
~
0.7 ± 0.2 1.3 + 0.2
3 4 5 6 7
0.8 + 0.2* 0.4 + 0.1* 0.2 + 0.1*
*Value is
significantly
group) treated with oxytocin days 3-7 of the oestrous cycle
4 per
=
on
Group
Group A Day of
goats (n
(group C)
Group
Testosterone (pg ml ])
Progesterone (ng ml I)
Testosterone (pg ml L)
Progesterone (ng ml )
55 ± 10 60 + 10 360 + 40* 520 + 60* 630 + 90*
0.8 ± 0.2 1.4 ±0.2 2.0 + 0.2 2.5 + 0.3 3.0 + 0.3
76+7 66+8 65 + 10 60+9 55+8
0.6 ± 0.2 0.9 + 0.2 1.8 + 0.2 2.4 + 0.3 2.9 + 0.3
-
~
~
~
C Testosterone (pg ml ') ~
66 + 62 ± 57 + 68 + 70 +
8
6 8 7 8
different from control (saline-treated) animals (P < 0.001).
All animals were observed for oestrus at least twice a day. Jugular vein blood (5 ml) was collected three times a day by venepuncture using 23-gauge needles. Blood was collected into heparinized tubes, centrifuged at 2000 g for 10 min and plasma 20°C until analysed. was stored at —
Results
Oxytocin administered to goats (group A) between day 3 and day 7 of the oestrous cycle induced luteal regression, indicated by a significant decrease in the progesterone concentration and
(P < 0.001), and with control saline-treated animals (group B) (Table 1). Co-administration of roIFN- to animals in group C completely blocked oxytocin-induced luteolysis and the rise in testosterone secretion (Table 1). Administration of roIFN- to goats at doses of 80 pg (group E), 160 pg (group F) and 320 pg (group G) between day 12 and day 18 of the oestrous cycle significantly (P < 0.01) delayed luteolysis and increased the duration of the cycle in a dosedependent manner to 23.2, 25.2 and 27.5 days in groups E, F and G, respectively, compared with 20 days for group D; roIFN- also significantly (P < 0.001) inhibited the rise in testosterone in animals in groups E, F and G compared with those in group D (Table 2). an
increase in the testosterone concentration
caused oestrus
Radioimmunoassay of hormones Plasma progesterone (0.1 ml) and testosterone (0.5 ml) were measured by radioimmunoassay, as described by Homeida (1986) and Homeida et al (1988). Progesterone antibody (provided by H. Dobson, Liverpool) was raised in rabbits against progesterone-11-succinyl-BSA, and used at a final dilution of 1:7000; crossreactions were 100% with progester¬ one and < 0.1% with corticosterone, desoxycorticosterone and ketocorticosterone. The intra-assay and interassay coefficients of variation for progesterone were 4.6% (« 25) and 11.6% (n = 20), respectively, for a plasma sample of low progesterone concentration (0.8 ng per tube) and 4.2% (n 25) and 11.6% (« 20), respectively, for a plasma sample of high concen¬ tration (5 ng per tube). The sensitivity of the assay was 48 pg per tube. Extraction efficiency was 85.1 ± 5% (mean + sd), and the results were corrected for extraction losses. Testosterone crossreactions obtained were 100% for tes¬ tosterone, 50% for dehydrotestosterone, 7% for androstanediol and < 0.02% for progesterone and oestradiol. The intra-assay coefficient of variation for testosterone was 11.9% (« 15) for a plasma sample of low testosterone concentration (60 pg per tube) and 12.9% (« 15) for a plasma sample of high testoster¬ one concentration (900 pg per tube), and the assay sensitivity was 13 pg per tube. The efficiency of radioactive hormone recovery was 75 ± 2% and values were corrected for extraction losses. =
compared
=
=
=
=
Statistical
analyses Data were expressed as means + sd. Analysis of variance (anova) for repeated measures using the general linear model (GLM) procedure of the statistical analysis system (SAS, 1985)
was
used to test the effect of saline, oxytocin or roIFN- . of means in different groups was made by
Comparison
Duncan's
multiple-range test.
Discussion of roIFN- (previously called ovine trophoblast protein-1) in this experiment rather than caprine trophoblast protein-1 was based on a number of similarities between them. Both are acidic proteins of low molecular masses, both have identical physical characteristics and are immunologically related (Gnatek el al, 1989). Intrauterine administration of roIFN- into the goat at the time of luteal regression delays luteolysis. Similarly, luteolysis is delayed in sheep and cattle following intrauterine infusion of ovine and bovine trophoblast interferons or conceptus protein (Knickerbocker et al, 1986; Vallet et al, 1988; Thatcher et al, 1989; Garverick et al, 1992). Co-administration of roIFN- and oxytocin also blocked oxytocin-induced luteolysis, giving further support to the hypothesis that roIFN- can behave as a
The
use
luteotrophic agent in goats.
and D), luteal regression In control animals (groups whether natural or induced was associated with a significant increase in testosterone secretion (Homeida and Cooke, 1984; Homeida, 1986; Homeida and Khalafalla, 1990), which was blocked by roIFN- . The exact mechanism whereby roIFNinhibits testosterone secretion is unknown. Serum testosterone
—
—
Table 2. Mean ( + sd)
D)
or
plasma concentration of progesterone and testosterone in goats («
recombinant ovine interferon tau at
Group Day of oestrous
cycle
Progesterone
(ngml-1) 4.6 ± 0.4 4.1 + 0.4 4.2 + 0.3 3.2 ± 0.4 2.2 + 0.2 1.6 + 0.2 0.6 + 0.1
12 13 14 15
16 17 18
*Value is
significantly
a
total dose of 80 pg (group E), 160 pg of the oestrous cycle
Testosterone
(pgml-1) 75 110 720 460 160 75 65
Progesterone
± 12 + 20 + 20 ± 80 + 20 + 10 + 10
(ngml-1) 4.3 4.3 4.5 4.8 4.2 4.1 4.2
± 0.3 + 0.3 + 0.3 ± 0.4 + 0.3* + 0.4* + 0.3*
Group
Testosterone
(pgml-1)
Progesterone
± 12 + 10 + 15* ± 10* 70+ 9* 60+9 75 + 10
4.6 4.5 4.3 4.6 4.7 4.3
Group
F
Testosterone
(ngml-1)
70 75 73 65
4 per group) treated with BSA (group 320 pg (group G) on days 12—18
(group F) and
Group E
D
=
Progesterone
(ngml-1)
(pgml-1)
± 0.3
± 10 + 12 + 10* ± 12* + 10* 75+9 65 + 10 72 75 68 65 60
+ 0.4 + 0.3 ± 0.3 + 0.3* + 0.3* 4.2 + 0.4*
4.1 4.4 4.5 4.7 4.2 4.3 4.2
± 0.3 + 0.4 + 0.3 ± 0.4 + 0.4* + 0.4* + 0.3*
G
Testosterone
(pgml-1) 80 ± 11 75 + 12
70+10* ± 9* + 10*
65 73 75 70
+ 10 + 10
different from control (P< 0.001).
concentrations in men decrease after injection of IFN- (Orava et al, 1986). Pretreatment with IFN- also reduces hCGstimulated testosterone secretion from cultured porcine Leydig cells (Orava, 1989). Systemic administration of IFN- to women reduces the concentration of oestradiol and progester¬ one in the blood (Kaupilla et al, 1982). Systemic effects of intrauterine roIFN- on the ovary can also be expected, since it has been reported that antiviral activity of ovine trophoblast-1 of the conceptus can occur in uterine venous serum, presum¬ ably after absorption from the uterine lumen (Schalue-Francis et al, 1991). The decrease in testosterone secretion induced by roIFN- could be a direct effect or it could occur via the inhibition of release of PGF2a, the uterine luteolysin (Horton and Poyser, 1976). Addition of PGF2(1 to incubations of corpora lutea increases testosterone synthesis (Shemesh et al, 1975). Testosterone and PGF2a are released on days 13 and 14 of the oestrous cycle of goats (Homeida and Cooke, 1982, 1984). Recombinant bovine and ovine interferons have been shown to decrease the release of uterine PGF2(1 in vitro (Barros et al, 1990; Ott et al, 1992) and in vivo (Plante et al, 1991; Ott et al, 1992).
Furthermore, bovine conceptus secretory protein induces prostaglandin inhibitor activity (Cross and Roberts, 1991). The effect of roIFN-
on
luteal function and testosterone
secretion in the
goat mimics that of an oxytocin antagonist (Homeida and Khalafalla, 1990). Both substances delay luteo¬
lysis and block natural and oxytocin-induced
testosterone
secretion that precedes oestrus. The oxytocin antagonist binds to oxytocin receptors inhibiting phospholipase A2, the arachidonic acid cascade and the production of prostaglandins (McCracken et al, 1984; Bazer et al, 1986; Homeida and Al-Eknah, 1992); roIFN- can also inhibit production of PGF2n (Ott et al, 1992) via inhibition of oxytocin receptor expression (Flint et al, 1992). However, the goats in groups A-G must have had functional oxytocin receptors (Roberts et al, 1976) at the time of interferon administration; the fact that roIFN- still prevented luteolysis suggests that it also has effects that do not involve the oxytocin receptor.
The authors thank the Director of the Experimental Station at King Faisal University for providing the goats and feed, F. Bazer (Center for
Animal
Biotechnology,
Texas A&M
University) for the gift of ovine
interferon, and H. Dobson (University of Liverpool) for the gift of antisera.
They
also thank A. E. Elbashir and A. Osman for technical
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