- Email: [email protected]
Oestradiol and the surge release of gonadotrophins in the ewe
Animal Reproduction Science, 34 (1994) 217-230
217
0378-4320/94/$07.00 © 1994 - Elsevier Science Publishers B.V. All rights reserved
Oestradiol and the surge release of gonadotrophins in the ewe I.B.J.K. Joseph 1, W.D. Currie 2, J.P. Ravindra, S.J. Cook, N.C. Rawlings* Department of Veterinary PhysiologicalSciences, Western Collegeof Veterinary Medicine, University of Saskatchewan, Saskatoon, Sask. STN OWO, Canada (Accepted 28 May 1993)
Abstract
This experiment was designed to study the effects of different doses and different patterns of change in circulating concentrations of oestradiol on the genesis of the surge release of luteinising hormone (LH) and follicle stimulating hormone (FSH), following progesterone withdrawal, in oestradiol and progesterone treated, long-term ovariectomised ewes. During the breeding season, 60 long-term ovariectomised ewes received progesterone releasing implants on Days 0, 2, 4, 6 and 10 of the study; two, one and two implants were then removed at 09: 00 h and 21 : 00 h on Day 11 and 09 : 00 h on Day 12, respectively. The ewes were also fitted with 0.5 cm or 6.0 cm oestradiol releasing implants for the duration of the study. The temporal profile of serum concentrations of progesterone closely resembled that of the luteal phase of the oestrous cycle of an intact ewe. With progesterone removal, several supplementary oestradiol treatments were applied, resulting in peak serum concentrations of oestrad iol similar to the luteal phase (basal) ( 6.6 + 1.2 pg m l - ~), follicular phase ( 17.54 _+2.6 pg m l - ~), or up to two times the peak follicular phase level seen in intact cyclic ewes. The mode of delivery of oestradiol varied from an additional implant or injection (i.m.), to intravenous infusion with increasing doses of oestradiol. Following progesterone withdrawal, gonadotrophin surges were observed in some, but not all of the ewes when serum concentrations of oestradiol were in the follicular phase range. No surges were seen when serum oestradiol concentrations were in the basal range. The pattern of increase in oestradiol following withdrawal of progesterone did not appear critical. Treatments resulting in constant high levels of oestradiol after progesterone withdrawal consistently elicited gonadotrophin surges, but the surge amplitudes were not as high as those resulting from rising serum concentrations of oestradiol. Peak serum oestradiol concentrations (28.7_+ 2.9 pg ml -~ ) in excess of follicular phase concentrations consistently resulted in surges, increased LH (hut not FSH) surge amplitude and decreased latency to the LH surge. Peak serum concentration of oestradiol of 36.4_ 4.1 pg ml-~ consistently resulted in surges but depressed LH and FSH surge amplitudes. Induction of FSH surges appeared to be less responsive to oestradiol than the induction of LH surges. *Corresponding author. Ipresent address: Department of Population Dynamics, Johns Hopkins University, Baltimore, M D 21205, USA. 2Present address: Department of Obstetrics and Gynaecology, Grace Hospital, University of British Columbia, BC, Canada.
SSD10378-4320(93)01249-8
218
LB.J.K. Joseph et al. / Animal Reproduction Science 34 (1994) 217-230
Introduction
Administration of oestradiol to ovariectomised or anoestrous ewes by various routes has been shown to result in a surge release of luteinising hormone (LH) (Goding et al., 1969; Radford et al., 1969; Karsch and Foster, 1975; Pant et al., 1977; Emons et al., 1986; Rozell and Keisler, 1990). The induced surge of LH secretion is caused by an increase in gonadotrophin releasing hormone (GnRH)secretion (Clarke and Cummins, 1985; Caraty et al., 1989; Moenter et al., 1990). Immunisation of the ewe against oestradiol blocks the preovulatory LH surge (Rawlings et al., 1978). At the end of the luteal phase of the oestrous cycle of the ewe, circulating concentrations of progesterone decline. This is accompanied by a rise in oestradiol secretion, which acts as a primary signal for the preovulatory gonadotrophin surge (Hauger et al., 1977; Pant et al., 1977). However, the preovulatory rise in the circulating concentration of oestradiol is often limited and no greater than changes seen in the luteal phase of the oestrous cycle (Pant et al., 1977; Rawlings et al., 1977 ). It would appear that the circulating concentrations of progesterone during the luteal phase of the oestrous cycle and the rate of disappearance of progesterone from the circulation at the end of the cycle, influence the timing but not the amplitude of the preovulatory LH and follicle stimulating hormone (FSH) surges (Jeffcoate et al., 1984; Haresign, 1985). In ovariectomised ewes treated with implants that constantly released progesterone and oestradiol, surges of both LH and FSH were observed when progesterone was withdrawn (Jeffcoate et al., 1984; Rawlings et al., 1984); the amplitude of these surges was on the low end of the normal range seen in intact ewes (Pant et al., 1977). In ovariectomised ewes, LH surges did not follow progesterone withdrawal, with serum oestradiol concentrations analogous to the luteal phase of an oestrous cycle, but if oestradiol was given after progesterone withdrawal to mimic levels seen in the follicular phase, surges resulted (Goodman et al., 1981; Fabre-Nys and Martin, 1991 ). The timing and amplitude of the LH surge appeared to be dependent on the dose and timing of administration of oestradiol after progesterone withdrawal; FSH was not monitored (Fabre-Nys and Martin, 1991; Goodman et al., 1981 ). The rate of increase of serum concentrations of oestradiol appeared to affect the timing and amplitude of the surge release of LH in ovariectomised ewes that had not been primed with progesterone (Rozell and Keisler, 1990). The purpose of the present study was to clarify the effect of dose and pattern of administration of oestradiol on the genesis of the surge release of both LH and FSH in ovariectomised ewes following progesterone withdrawal.
LB.J.K. Joseph et al. / Animal Reproduction Science 34 (1994) 217-230
219
Materials and methods
Experimental animals Sixty sexually mature crossbred ewes were kept outside in dry lots. During experiments the ewes were housed indoors under fluorescent lighting, with artificial daylength set to simulate natural daylength. The ewes were fed alfalfa pellets and alfalfa-brome hay, with cobalt-iodised salt and water freely available. Ewes were ovariectomised early in the breeding season and rested for at least 3-4 weeks before being used in this study.
Implants and infusions The steroid implants were prepared by mixing 10% (w/w) steroid with liquid silicone elastomer (RTV Silicone Elastomer, Factor II, Lakeside, AZ) and a curing catalyst. The oestradiol-17fl elastomer mixture was injected into silastic tubing of 0.32 cm inner diameter (i.d.) and 0.48 cm outer diameter (o.d.) (Dow Coming, Midland, MI ). When cured, the implants were cut into 0.5 or 6.0 cm lengths. The progesterone elastomer mixture was injected into tygon tubing moulds (Norton Plastics, Okbon, OH) of 0.48 cm i.d. The implants were removed from the moulds after 24 h and cut into 10 cm lengths. The implants were incubated in sterile 0.9% (w/v) saline for 24 h, at 37 °C, prior to insertion. Implants were introduced subcutaneously in the axillary region under local anaesthesia (Duracaine, Rogar/STB Inc., Montreal, Que., Canada). The release pattern of steroid from the implants has been described previously (Jeffcoate et al., 1984; Rawlings et al., 1984; Joseph et al., 1992). For infusions and collection of blood, ewes were catheterised in both jugular veins with vinyl tubing ( 1.00 mm i.d., 1.50 mm o.d.; SV-70, Dural Plastics and Engineering, Dural, N.S.W., Australia). Infusions were carded out using a ten-channel peristaltic pump (Manostat, New York), connected to the right jugular vein via silicone rubber tubing (2.38 mm i.d., 3.97 mm o.d. ).
Experimental protocol All ewes received a progesterone implant on Days 0, 2, 4, 6 and 10 of the study. Two, one and two progesterone implants were removed at 09: 00 h and 21:00 h on Day 11 and 09:00 h on Day 12, respectively (Fig. 1 ). Oestradiol implants were also inserted on Day 0 of the study. Forty-eight ewes received an oestradiol implant which was 0.5 cm in length, and 12 ewes received an implant 6 cm in length (Table 1 ). The oestradiol implants were left in place for the duration of the experiment. When progesterone implants were removed, additional treatments with oestradiol were imposed; these are summarised in Table 1 and Fig. 2. Twenty-four and six ewes treated respectively
220
I.B.J.K. Joseph et al. / Animal Reproduction Science 34 (1994) 217-230
6.0-
5.0-
4.0-
3.o 2.0
1.0
0.0
-
-2
|
0
.
1
2
•
i
4
•
i
.
6
i
8
•
i
10
-
i
12
•
=
-
14
Day Fig. 1. The temporal pattern of serum concentrations of progesterone (.(+ SEM) in long-term ovariectomised ewes given a 10 cm × 0.48 cm solid silastic rod containing progesterone on Days 0, 2, 4, 6 and 10 (J,). Two, one and two progesterone implants were removed at 09:00 h and 21 : 00 h on Day 11 and 09: 00 h on Day 12, respectively (T).
with 0.5 cm and 6 cm oestradiol implants were infused with oestradiol in saline for 40 h from 22:00 h on Day 11 (1 h after the second removal of progesterone implants) to 14: 00 h on Day 13. In 12 ewes treated with 0.5 cm oestradiol implants, the strength of infusate was doubled every 5 h, starting from 0.01/zg h - 1, to a final dose of 1.5 #g h - ~given in the last 5 h (low dose oestradiol infusion, Table 1 ). In all other ewes infused with oestradiol, the strength of infusate was doubled every 5 h, starting form 0.02/zg h - ~and with 3.0/zg h -~ infused in the last 5 h (high dose oestradiol infusion, Table 1 ). Two groups of six ewes treated with either 0.5 or 6 cm oestradiol implants, were infused with saline for 40 h from 22: 00 h on Day 11 to 14: 00 h on Day 13 (control, Table 1 ). In one group of ewes treated with 0.5 cm oestradiol implants, 10/zg of oestradiol in oil was injected (i.m.) 24 h after the last progesterone implant was removed (09:00 h on Day 13, injection; see Table 1 and Fig. 2). Finally, in one group of ewes treated with 0.5 cm oestradiol implants, one additional 0.5 cm oestradiol implant was introduced 12 h after the last progesterone implant was removed (21:00 h on Day 12, implant; see Table 1 and Fig. 2 ).
Blood sampling and analysis Daily blood samples were taken throughout the study for estimation of progestcrone concentration. Hourly samples were collected from 10: 00 h on Day 11 ( 1 h after onset of removal of progesterone implants) to 22: 00 h on Day
I.B.J.K. Joseph et al. /Animal Reproduction Science 34 (1994) 217-230
"0
0
0
~
. . . . .
ox.~
--i ~.
.o~ "= d
"~
~.~ 0
~.~ r~l
~-~
0
~
+l+l+l+l+l+l
0
+1'~
o ~ ,~ ~ .-. ~
e
~ +1+1+1+1+1+1
!e: N ~ ua'~ +~
+I+I+I+I+I+I
,S
~
c5 V ¢.)
+1+1+1+1+1+1
~e ~N
•~
o
o
0
.o
Oo~-~
e-t ~ N e~ ~ 0
~.~
o ~
0.~
0
~
~.~
o
221
401
222
I.B.J.K. Joseph et al. / Animal Reproduction Science 34 (1994) 217-230
3o
"6
~
20
L~111 •
10 •
•
0
4O
73o
i
•
•
•
i
24
•
•
•
|
48
•
"
"
0
72
•
•
•
0
i
•
•
•
24
|
•
•
•
I
48
"
'
'
72
40-
C
V
3O
-6 20
lO
•
•
•
i 24
•
•
•
i
•
•
•
48
i
•
•
•
0
•
.
.
i
•
.
•
•
•
i
•
24
Time after
•
•
|
48
•
•
.
i 48
°1o°l 0
•
24
72
i
•
•
.
.
•
i 72
.
.
•
i
96
•
72
Progesterone (h)
Fig. 2. The temporal pattern of serum concentrations of oestradiol ( X + SEM) in long-term ovariectomised ewes treated with oestradiol and progesterone releasing implants. Oestradiol implants 0.5 cm×0.48 cm ( a ) - ( d ) , or 6.0 cm×0.48 cm (e) were inserted on Day 0 and progesterone implants on Days 0, 2, 4, 6 and 10. Two, one and two progesterone implants were removed at 09: 00 h and 21 : 00 h on Day 11 and 09: 00 h on Day 12, respectively• Serum oestradiol concentrations are shown for 72 h following the first removal of progesterone implants (09:00 h on Day 11 to 09:00 h on Day 14). Over this period of time, additional oestradiol treatments were given as follows: (a) low dose or (b), (e) high dose infusion from 22:00 h on Day 11 until 14:00 h on Day 13 (0.5 cm implant+low dose infusion (F--t); 0.5 cm implant+high dose infusion (~- -1); 6.0 cm implant+high dose infusion (~- -t); (c) injection ( i.m. ) at 09: 00 h on Day 13 (0.5 cm implant + injection ( ~ ) ) or (d) an extra 0.5 cm × 0.48 cm implant at 21 : 00 h on Day 12 (0.5 cm implant + 0.5 cm implant ( ~ ) ). Further details of oestradiol doses are given in the text•
15, to characterise the surge release of gonadotrophins. Every second sample was analysed for concentrations of oestradiol. On Days 8 and 12, blood samples were taken every 10 min for 6 h to characterise pulsatile LH secretion.
L B.J.K. Joseph et al. /Animal Reproduction Science 34 (1994) 217-230
223
The sampling on Day 12 started 1 h after the last progesterone implant was removed.
Hormone assays Serum concentrations of FSH, LH, progesterone and oestradiol were determined in validated radioimmunoassays (Currie and Rawlings, 1989; Joseph et al., 1992). Concentrations of FSH and LH are expressed in terms of NIDDK-oFSH-RP 1 and NIDDK-oLH-24, respectively. The sensitivity of the gonadotrophin and steroid assays is expressed as the lowest standard different from zero, using an unpaired t-test. The limits of detection were 0.25 ng m l - 1 and 0.06 ng m l - ~ for FSH and LH respectively. Intra- and inter-assay coefficients of variation (CVs) for ovine reference sera with mean FSH concentrations of 14.1 ng m l - 1 or 1.13 ng m1-1 were 6.6% ( n = 8 ) and 9.6% ( n = l l ) or 7.6% ( n = l l ) and 9.7% (n = 110), respectively. Intra- and inter-assay CVs for ovine reference sera with a mean LH concentration of 3.01 ng ml -~ or 0.15 ng m1-1 were 5.0% ( n = 18) and 9.9% ( n = 110) or 9.6% ( n = 18) and 14.9% ( n = 110), respectively. The limits of detection for the oestradiol and progesterone assays were 1 pg ml-1 and 50 pg ml-1, respectively. For sera with oestradiol concentrations of 23 pg m1-1, 11.3 pg ml -~ or 8.8 pg ml -~, intra- and inter-assay CVs were 7.3% ( n = 2 ) and 9.1% ( n = 1 0 ) , 11% ( n = 2 ) and 11.6% ( n = 1 0 ) or 9.4% ( n = 2 ) and 12.5% ( n = 11 ), respectively. For sera with progesterone concentrations of 1.07 ng ml-~ or 2.15 ng ml-1, intra- and inter-assay CVs were 9.1% ( n = 7 ) and 11.4% ( n = 4 2 ) or 6.8% ( n = 7 ) and 9.5% ( n = 4 0 ) , respectively.
Statistical analysis The PULSAR program (Merriam and Wachter, 1982) was used to identify LH pulse frequency (pulses h - 1), amplitude (ng m l - ~), basal serum LH and mean serum LH concentrations (ng m l - 1 serum ) in blood samples taken every 10 min for 6 h on Days 8 and 12 (basal serum LH values are those after removal of pulses). The values of the G parameters used were 4.40, 2.60, 1.92, 1.46 and 1.13 for parameters 1, 2, 3, 4 and 5, respectively. The values assigned to the Baxter parameters were 0, 0.39 and 1.71 for A, B and C, respectively. According to this method, serum FSH concentrations did not show evidence of pulsatility. The surges of LH and FSH were identified visually. Data are presented as means and standard errors of the mean. Data were analysed by one-way and two-way analysis of variance as appropriate. Repeated measures analysis of variance was employed for the hormonal data resulting from blood samples collected every 10 min for 6 h on Days 8 and
224
LB.J.K. Joseph et al. /Animal Reproduction Science 34 (1994.) 217-230
12. After analysis of variance, individual treatment effects were examined by test of least significant differences.
Results
Serum steroids concentration: temporal patterns The temporal pattern of serum concentrations of progesterone produced by the progesterone implant regimen are shown in Fig. 1. The peak concentrations of oestradiol produced by the various treatments applied after progesterone withdrawal are shown in Table 1. The patterns of oestradiol produced by the various treatments are shown in Fig. 2. Serum progesterone concentration rose up to Day 8, and thereafter declined as implants were removed on Days 11 and 12. Peak progesterone concentrations varied quite markedly amongst ewes, as did the timing of the peak (Fig. 1 ). Serum concentrations of oestradiol produced by the two sizes of implants (0.5 cm × 0.48 cm vs. 6.0 cm × 0.48 cm) differed significantly ( P < 0.05 ) (Table 1 ). Infusion of oestradiol at either low or high dose produced peak levels of oestradiol that differed and were greater than those in control, non-infused ewes ( P < 0.05). Peak oestradiol concentration in ewes with small or large oestradiol implants (0.5 cm vs. 6 cm implants) did not differ when infused with the high dose of oestradiol ( P > 0.05 ). The peak concentration of oestradiol created by either injecting oestradiol or adding an extra 0.5 cm implant, after progesterone withdrawal, to ewes with the 0.5 cm implant were similar to ewes with a 0.5 cm implant and receiving the low dose infusion or ewes with the 6 cm oestradiol implant alone (Table 1 ). Following progesterone withdrawal, serum oestradiol concentrations in ewes treated with only one oestradiol implant were fairly constant. Infusion of oestradiol produced a gradual rise in oestradiol concentrations over the period of infusion (Fig. 2 ). Injection of oestradiol or insertion of a second oestradiol implant, produced a short-lived peak in concentration of oestradiol (less than 3 h), followed by a rapid decline. Following the peak, oestradiol levels were maintained at a higher level in ewes with two 0.5 cm oestradiol implants compared with those with only one (Fig. 2).
Gonadotrophin surges No gonadotrophin surges were seen following progesterone withdrawal in the presence of one 0.5 cm oestradiol implant (Table 1 ). In the other groups of ewes treated with 0.5 cm implants, gonadotrophin surges were seen in 11 / 12, 3/6 and 4/6 of the ewes receiving the high dose infusion, injection or added implant; in the low dose infusion group, 6/12 ewes had LH surges but only 1/12 had an FSH surge. In ewes treated with 6 cm implants, 5/6 had
LB.J.K. Joseph et al. / Animal Reproduction Science 34 (1994) 217-230
225
Table 2 Mean and basal serum concentrations o f L H and LH pulse amplitude and frequency in long-term ovariectomised ewes treated with progesterone releasing implants for 12 days and fitted with 0.5 c m × 0 . 4 8 cm or 6.0 c m × 0 . 4 8 cm oestradiol implants. Blood samples were taken every 10 min for 6 h on Days 8 and 12 o f treatment. The last progesterone implant was removed 1 h before sampling on Day 12. With progressterone withdrawal, three groups of ewes received either low or high dose infusions of oestradiol starting at 22:00 h on Day 11. See text and Fig. 1 for details of treatments Length of oestradiol implant (cm)
Post-progesterone Serum LH concentration oestradiol (ng ml- 1) treatment Mean Basal
LH pulse
Frequency (pulse h -1 )
Amplitude (ngm1-1 )
Day 8 0.5 6.0
0.35+0.07 b 0.17+0.01 ~
0.17+0.03 ~ 0.10+0.00 ~
0.56+0.11 a 0.61 +0.08 ~
0.84+0.22 b 0.42+0.06 ~
0 . 7 4 + 0 . 1 0 °a 0 . 7 8 + 0 . 1 5 *~b 1.31 + 0 . 2 6 *b 1.06 + 0.02 *b 0.92 + 0.02 *a
0 . 4 9 + 0 . 0 6 *ab 0 . 4 6 + 0 . 0 9 *ab 1.00+0.21 °c 0 . 5 4 + 0.02 °b 0.40 + 0.03 °~
1.23+0.12 *ab 0 . 9 7 + 0 . 1 4 *~ 1.35+0.13 *b 1.51 + 0 . 0 6 *b 1.36 + 0.07 *b
0.63+0.09 a 0 . 8 4 + 0 . 1 7 ab 0.79+0.09 a 1.12+0.05 °b~ 1.20 + 0.07 *c
Day 12 0.5 0.5 0.5 6.0 6.0
Control Low dose High dose Control High dose
*Significant differences between Day 8 and Day 12 ( P < 0.05).
Different superscripts within columns indicate signifcant differences between treatments for Day 8 and Day 12 separately ( P < 0.05 ).
gonadotrophin surges when no other oestradiol treatment was given and 6/6 had gonadotrophin surges when a high dose infusion was given. LH surges of similar amplitude followed progesterone withdrawal in ewes with 0.5 cm oestradiol implants and receiving the low dose infusion, injection or extra oestradiol implant and in ewes with one 6 cm oestradiol implant alone or with additional high dose infusion (Table 1 ). The ewes treated with a single 0.5 cm oestradiol implant and high dose infusion had LH surges of the greatest amplitude (Table 1 ) ( P < 0 . 0 5 ) . Latency from progesterone withdrawal (removal of first implant) to the peak of the LH surge was shorter in ewes receiving the high dose oestradiol infusion compared with all other ewes (Table 1 ) ( P < 0 . 0 5 ) . Unlike LH, the amplitudes of FSH surges were lower in all ewes with 6 cm implants than in those with 0.5 cm implants, with the exception of the one ewe in the low dose infusion group that had a surge. High dose infusion resulted in a mean FSH surge amplitude of 19.5_ 1.5 ng m l - l in ewes with 0.5 cm oestradiol implants, compared with 8.7 _+0.0 ng ml-~ in the one ewe having an FSH surge in the low dose infusion group. Again, unlike LH, the high dose infusion of oestradiol did not significantly reduce the latency from progesterone withdrawal to the FSH surge peak (Table 1 )
(P>0.05).
226
t.B.J.K. Joseph et al. /Animal Reproduction Science 34 (1994) 217-230
Pulsatile secretion of LH Pulsatile secretion of LH was examined on Days 8 and 12 of the experiment (Table 2). During progesterone and oestradiol treatment on Day 8, mean serum concentrations of LH and LH pulse amplitude were lower in ewes with 6 cm oestradiol implants than in those with 0.5 cm implants. Following progesterone withdrawal, mean and basal serum concentrations of LH and LH pulse frequency increased but LH pulse amplitude only increased in ewes treated with the larger (6 cm) oestradiol implant. On Day 12 (after progesterone withdrawal), mean and basal serum concentrations of LH tended to be higher in ewes with 6 cm oestradiol implants alone or 0.5 cm implants and the high dose oestradiol infusion; LH pulse frequency was lowest in ewes with 0.5 cm implants and low dose oestradiol infusion.
Discussion The progesterone concentrations resulting from implant treatments in this study were very similar to those seen in normal oestrous cycles in the ewe, both in pattern and absolute values (Rawlings et al., 1977). The basal serum oestradiol concentrations resulting from one small implant (0.5 cm) in our results were similar to concentrations we have seen in the luteal phase of normal cyclic ewes (4.8 +_0.1 pg ml -~ ) (Rawlings and Cook, 1992). The peak oestradiol concentrations resulting from two small implants, one large implant (6 cm ), low dose infusion of oestradiol or injection of oestradiol, were in the upper end of the range for the follicular phase of normal cyclic ewes ( 12.7 _+3.3 pg ml- ~) (Rawlings and Cook, 1992 ). Oestradiol concentrations resulting from high dose infusions were almost twice peak follicular phase levels. In previous studies with progesterone/oestradiol treated ovaricctomiscd ewes, basal concentrations of oestradiol like those of the luteal phase of cyclic ewes, did not result in LH surges following progesterone withdrawal (Goodman et al., 1981 ). However, if additional oestradiol was given after progesterone withdrawal to raise serum concentrations of oestradiol to those in the follicular phase of the oestrous cycle, LH surges were seen (Goodman et al., 1981; Fabrc-Nys and Martin, 1991 ). In addition, if serum concentrations of oestradiol were high but constant in oestradiol/progesterone treated ovaricctomised ewes, LH and FSH surges resulted from the withdrawal of progesterone (Jeffcoate et al., 1984; Rawlings et al., 1984). In our present work, progesterone withdrawal against constant basal serum concentrations of oestradiol, as expected (6.6_+ 1.2 pg ml-~; Table 1 ), did not result in gonadotrophin surges. LH and FSH surges were seen with all oestradiol treatments that resulted in peak serum concentrations of oestradiol that were within or greater than the range of values for the follicular phase of the oestrous cycle
LB.J.K. Joseph et aL / Animal Reproduction Science 34 (1994) 217-230
227
of the ewe ( 15-19 pg m l - ~; Table 1 ), regardless of whether oestradiol levels were constant or rising. LH surge amplitudes did not differ amongst these treatment groups, but not all ewes responded. This would suggest that these oestradiol concentrations were around threshold for the induction of gonadotrophin surges. Therefore, it would appear that an absolute level of oestradiol is required to cause a gonadotrophin surge following progesterone withdrawal, but that a particular pattern of increase is not important. In the present study, it appeared that LH surge amplitude was low in response to two treatments, the low dose oestradiol infusion into ewes with 0.5 cm oestradiol implants and ewes fitted with 6.0 cm oestradiol implants alone, even though follicular phase oestradiol concentrations were seen. These apparent differences were not significant for LH but they were for FSH; in fact, for FSH the former treatment resulted in a surge in only one ewe. It is difficult to explain why the low dose oestradiol infusion was ineffective in causing FSH surges. The 6.0 cm oestradiol implant alone resulted in FSH and LH surges in a fairly consistent manner but the surge amplitudes were low. Low surge amplitudes have been noted previously following progesterone withdrawal in oestradiol/progesterone treated ovariectomised ewes, when serum oestradiol concentrations were high but constant (Jeffcoate et al., 1984; Rawlings et al., 1984 ). Clearly, progesterone withdrawal and some degree of increase in serum concentrations of oestradiol are required to optimally induce the preovulatory gonadotrophin surge. In the present experiment, when the high dose infusion of oestradiol was given following progesterone withdrawal, LH and FSH surges were consistently produced. In addition, in ewes with 0.5 cm oestradiol implants and high dose oestradiol infusion, LH surge amplitude was higher than all other treatments and in fact, higher than in most intact ewes (Rawlings and Cook, 1992). It was of interest that the high peak serum oestradiol concentration produced by the high dose infusion in ewes with 0.5 cm oestradiol implants, in our work, did not cause extremely high FSH surge amplitudes as it did for LH. There is certainly an indication in our data that the FSH surge is less responsive to the oestradiol positive feedback process. The high dose infusion given to ewes with the large 6.0 cm oestradiol implant depressed LH and particularly FSH surge amplitudes. This apparent dose dependency for induction of LH surges and amplitude of the LH surge, with a depression at the highest serum oestradiol concentrations, has been indicated previously (Goodman et al., 1981; Rawlings et al., 1984). In general, in our work, the higher the peak serum concentration of oestradiol produced in ovariectomised ewes after progesterone withdrawal, the shorter the latency to the LH surge; this was not as clear for FSH. This effect has been seen previously for LH (Karsch et al., 1980; Goodman et al., 1981; Fabre-Nys and Martin, 1991 ). It has also been previously suggested that, in ovariectomised ewes with no progesterone priming, the rate of increase in serum concentrations of oestradiol influences the timing of the LH surge
228
LB.J.K. Joseph et al. /Animal Reproduction Science 34 (1994) 217-230
(Rozell and Keisler, 1990). We saw little evidence for this in our study. FabreNys and Martin ( 1991 ) have also shown that the timing of the increase in serum levels of oestradiol after progesterone withdrawal influences LH surge amplitude and timing in progesterone-primed ovariectomised ewes. The serum concentrations of oestradiol required to consistently elicit gonadotrophin surges in our work tended to be high compared with peak serum concentrations of oestradiol in intact ewes (Rawlings and Cook, 1992). However, the animals used in the present study were long-term ovariectomised ewes. It has been shown that negative feedback suppression ofgonadotrophin secretion by both progesterone and oestradiol decreases with time in the ovariectomised ewe (Karsch et al., 1977; Joseph et al., 1992); it is possible, therefore, that positive feedback sensitivity to physiological levels of oestradiol may also change with time. It is of interest that the basal or lowest serum oestradiol concentrations observed in the present experiment were similar to those that appeared to be at a threshold for the negative feedback regulation of LH secretion during the breeding season in long-term ovariectomised ewes (Joseph et al., 1992). In the present work, these serum concentrations ofoestradiol did not cause gonadotrophin surges but some of the higher concentrations of oestradiol did, indicating a clear differentiation between the threshold for the negative and positive feedback effects of oestradiol on gonadotrophin secretion. Observations of pulsatile LH secretion on Days 8 and 12 produced results that were largely as expected (Goodman and Karsch, 1980; Rawlings et al., 1984; Joseph and Rawlings, 1990). Progesterone largely suppresses LH pulse frequency and oestradiol LH pulse amplitude (Goodman and Karsch, 1980). On Day 8 the larger oestradiol implant resulted in a greater suppression of LH pulse amplitude and mean serum concentration of LH than the small implant (Table 2). As expected, LH pulse frequency rose by Day 12 (after progesterone removal) (Karsch et al., 1983 ). Interestingly, basal serum concentrations of LH also rose and together with the frequency effect this resulted in an increase in mean serum LH concentrations. On Day 12 there was some tendency toward variation between treatments in parameters of pulsatile LH secretion, but this was not consistent (Table 2). This could have been because the oestradiol infusions had only been running for 11 h and were still at fairly low doses by the time the intensive blood sampling was done. The increased LH pulse amplitude on Day 12 in ewes with 6.0 cm oestradiol implants was not expected and is difficult to explain. In conclusion, following progesterone removal in long-term ovariectomised ewes treated with oestradiol and progesterone releasing implants, gonadotrophin surges resulted in some but not all ewes when serum concentrations of oestradiol were in the range seen in intact ewes in the follicular phase of an oestrous cycle. The pattern of increase in oestradiol did not appear critical for the initiation of a gonadotrophin surge. Although constant high levels
LB.J.K. Joseph et al. / AnimaI Reproduction Science 34 (1994) 217-230
229
of oestradiol consistently elicited gonadotrophin surges they did not give surges of as great an amplitude as those arising from rising serum concentrations of oestradiol. Increasing serum concentrations of oestradiol above follicular phase levels consistently induced gonadotrophin surges, increased LH but not FSH surge amplitude and decreased latency to the LH surge. The highest peak serum concentrations of oestradiol used in this study actually depressed LH and FSH surge amplitude. The mechanism leading to the FSH surge may be somewhat less responsive to oestradiol than that leading to the LH surge. Acknowledgements The authors thank M. Buckley and her staff for animal care. Gonadotrophin assay reagents were obtained from NIDDK. I.B.J.K. Joseph was a recipient of a Canadian Commonwealth Scholarship. This work was supported by NSERC, Canada.
References Caraty, A., Locatelli, A. and Martin, G.B., 1989. Biphasic response in the secretion of gonadotrophin-releasing hormone in ovariectomised ewes injected with oestradiol. J. Endocrinol., 123: 375-382. Clarke, I.J. and Cummins, J.T., 1985. Increased gonadotropin-releasing hormone pulse frequency associated with estrogen-induced luteinizing hormone surges in ovariectomised ewes. Endocrinology, 116: 2376-2383. Currie, W.D. and Rawlings, N.C., 1989. Fluctuations in responsiveness of LH and lack of responsiveness of FSH to prolonged infusion of morphine and naloxone in the ewe. J. Reprod. Fertil., 86: 359-366. Emons, G., Schuppe, H., Peter, M., Brack, C. and Ball, P., 1986. Modulation of LH secretion in ovariectomized ewes by constant infusions of estradiol and 4-hydroxyoestradiol--effects of varying infusion time and of high estrogen doses. Acta Endocrinol., 113:219-225. Fabre-Nys, C. and Martin, G.B., 1991. Roles of progesterone and oestradiol in determining the temporal sequence and quantitative expression of sexual receptivity and the preovulatory LH surge in the ewe. J. Endocrinol., 130: 367-379. Goding, J.R., Cart, K.J., Brown, J.M., Kaltenback, C.C., Cumming, I.A. and Mole, B.J., 1969. Radioimmunoassay for ovine luteinising hormone. Secretion of luteinizing hormone during estrus and following estrogen administration in the sheep. Endocrinology, 85:133-142. Goodman, R.L. and Karsch, F.J., 1980. Pulsatile secretion of luteinizing hormone; differential suppression by ovarian steroids. Endocrinology, 107:1286-1290. Goodman, R.L., Legan, S.J., Ryan, K.D., Foster, D.L. and Karsch, F.J., 1981. Importance of variation in behavioral and feedback action of oestradiol to the control of seasonal breeding in the ewe. J. Endocrinol., 89: 229-240. Haresign, W., 1985. Comparison of the rate of decline in plasma progesterone concentration at a natural and progesterone-synchronized oestrus and its effect on tonic LH secretion in the ewe. J. Reprod. Fertil., 75: 231-236. Hauger, R.L., Karsch, F.J. and Foster, D.L., 1977. A new concept for control of the oestrous cycle of the ewe. Based on the temporal relationships between luteinizing hormone, oestra-
2 30
LB.J.K. Joseph et al./Animal Reproduction Science 34 (1994) 217-230
diol and progesterone in peripheral serum and evidence that progesterone inhibits tonic LH secretion. Endocrinology, 101: 807-818. Jeffcoate, I.A., Rawlings, N.C. and Rieger, D., 1984. Progesterone and the surge release of gonadotropins in the ewe. Domest. Anim. Endocrinol., 1:309-317. Joseph, I.B.J.K. and Rawlings, N.C., 1990. Infusion of estradiol during the luteal phase of the estrous cycle in the ewe completely eliminates LH pulses. Can. J. Anim. Sci., 70:1147-1150. Joseph, I.B.J.K., Currie, W.D. and Rawlings, N.C., 1992. Luteinising hormone and follicle stimulating hormone secretion in ovariectomised ewes: Effects of time post ovariectomy, season and oestradiol. J. Reprod. Fertil., 94:511-523. Karsch, F.J. and Foster, D.L., 1975. Sexual differentiation of the mechanism controlling the preovulatory discharge of luteinising hormone in sheep. Endocrinology, 97: 373-382. Karsch, F.J., Legan, S.J., Hauger, R.L. and Foster, D.L., 1977. Negative feedback action of progesterone on tonic luteinising hormone secretion in the ewe: Dependence on the ovaries. Endocrinology, 101: 800-806. Karsch, F.J., Legan, S.J., Ryan, K.D. and Foster, D.L., 1980. Importance of estradiol and progesterone in regulating LH secretion and estrous behaviour during the sheep estrous cycle. Biol. Reprod., 23: 404-413. Karsch, F.J., Foster, D.L., Bittman, E.L. and Goodman, R.L., 1983. A role for estradiol in enhancing luteinising hormone pulse frequency during the follicular phase of the estrous cycle of sheep. Endocrinology, 113:1333-1359. Merriam, G.R. and Wachter, K.W., 1982. Algorithms for the study of episodic hormone secretion. Am. J. Physiol., 243:E310-E318. Moenter, S.M., Caraty, A. and Karsch, F.J., 1990. The estradiol-induced surge ofgonadotropinreleasing hormone in the ewe. Endocrinology, 127:1375-1384. Pant, H.C., Hopkinson, C.R.N. and Fitzpatrick, R.J., 1977. Concentrations of oestradiol, progesterone, luteinizing hormone and follicle-stimulating hormone in the jugular venous plasma of ewes during the oestrous cycle. J. Endocrinol., 773: 247-255. Radford, H.M., Wheatley, I.S. and Wallace, A.L.C., 1969. The effects of oestradiol benzoate and progesterone on secretion of luteinizing hormone in the ovariectomised ewe. J. Endocrinol., 44: 135-136. Rawlings, N.C. and Cook, S.J., 1992. LH secretion around the time of the preovulatory gonadotrophin surge in the ewe. Anim. Reprod. Sci., 30: 289-299. Rawlings, N.C., Kennedy, S.W., Chang, C.H., Hill, J.R. and Henricks, D.M., 1977. Onset of seasonal anestrus in the ewe. J. Anita. Sci., 44: 791-797. Rawlings, N.C., Kennedy, S.W. and Henricks, D.M., 1978. Effect of active immunisation of the cyclic ewe against oestradiol-17ft. J. Endocrinol., 76: I 1-19. Rawlings, N.C., Jeffcoate, I.A. and Rieger, D.L., 1984. The influence of oestradiol-17fl and progesterone on peripheral serum concentration of luteinizing hormone and follicle stimulating hormone in the ovariectomized ewe. Theriogenology, 22: 473-488. Rozell, T.G. and Keisler, D.H., 1990. Effects of oestradiol on LH, FSH and prolactin in ovariectomized ewes. J. Reprod. Fertil., 88: 645-653.
217
0378-4320/94/$07.00 © 1994 - Elsevier Science Publishers B.V. All rights reserved
Oestradiol and the surge release of gonadotrophins in the ewe I.B.J.K. Joseph 1, W.D. Currie 2, J.P. Ravindra, S.J. Cook, N.C. Rawlings* Department of Veterinary PhysiologicalSciences, Western Collegeof Veterinary Medicine, University of Saskatchewan, Saskatoon, Sask. STN OWO, Canada (Accepted 28 May 1993)
Abstract
This experiment was designed to study the effects of different doses and different patterns of change in circulating concentrations of oestradiol on the genesis of the surge release of luteinising hormone (LH) and follicle stimulating hormone (FSH), following progesterone withdrawal, in oestradiol and progesterone treated, long-term ovariectomised ewes. During the breeding season, 60 long-term ovariectomised ewes received progesterone releasing implants on Days 0, 2, 4, 6 and 10 of the study; two, one and two implants were then removed at 09: 00 h and 21 : 00 h on Day 11 and 09 : 00 h on Day 12, respectively. The ewes were also fitted with 0.5 cm or 6.0 cm oestradiol releasing implants for the duration of the study. The temporal profile of serum concentrations of progesterone closely resembled that of the luteal phase of the oestrous cycle of an intact ewe. With progesterone removal, several supplementary oestradiol treatments were applied, resulting in peak serum concentrations of oestrad iol similar to the luteal phase (basal) ( 6.6 + 1.2 pg m l - ~), follicular phase ( 17.54 _+2.6 pg m l - ~), or up to two times the peak follicular phase level seen in intact cyclic ewes. The mode of delivery of oestradiol varied from an additional implant or injection (i.m.), to intravenous infusion with increasing doses of oestradiol. Following progesterone withdrawal, gonadotrophin surges were observed in some, but not all of the ewes when serum concentrations of oestradiol were in the follicular phase range. No surges were seen when serum oestradiol concentrations were in the basal range. The pattern of increase in oestradiol following withdrawal of progesterone did not appear critical. Treatments resulting in constant high levels of oestradiol after progesterone withdrawal consistently elicited gonadotrophin surges, but the surge amplitudes were not as high as those resulting from rising serum concentrations of oestradiol. Peak serum oestradiol concentrations (28.7_+ 2.9 pg ml -~ ) in excess of follicular phase concentrations consistently resulted in surges, increased LH (hut not FSH) surge amplitude and decreased latency to the LH surge. Peak serum concentration of oestradiol of 36.4_ 4.1 pg ml-~ consistently resulted in surges but depressed LH and FSH surge amplitudes. Induction of FSH surges appeared to be less responsive to oestradiol than the induction of LH surges. *Corresponding author. Ipresent address: Department of Population Dynamics, Johns Hopkins University, Baltimore, M D 21205, USA. 2Present address: Department of Obstetrics and Gynaecology, Grace Hospital, University of British Columbia, BC, Canada.
SSD10378-4320(93)01249-8
218
LB.J.K. Joseph et al. / Animal Reproduction Science 34 (1994) 217-230
Introduction
Administration of oestradiol to ovariectomised or anoestrous ewes by various routes has been shown to result in a surge release of luteinising hormone (LH) (Goding et al., 1969; Radford et al., 1969; Karsch and Foster, 1975; Pant et al., 1977; Emons et al., 1986; Rozell and Keisler, 1990). The induced surge of LH secretion is caused by an increase in gonadotrophin releasing hormone (GnRH)secretion (Clarke and Cummins, 1985; Caraty et al., 1989; Moenter et al., 1990). Immunisation of the ewe against oestradiol blocks the preovulatory LH surge (Rawlings et al., 1978). At the end of the luteal phase of the oestrous cycle of the ewe, circulating concentrations of progesterone decline. This is accompanied by a rise in oestradiol secretion, which acts as a primary signal for the preovulatory gonadotrophin surge (Hauger et al., 1977; Pant et al., 1977). However, the preovulatory rise in the circulating concentration of oestradiol is often limited and no greater than changes seen in the luteal phase of the oestrous cycle (Pant et al., 1977; Rawlings et al., 1977 ). It would appear that the circulating concentrations of progesterone during the luteal phase of the oestrous cycle and the rate of disappearance of progesterone from the circulation at the end of the cycle, influence the timing but not the amplitude of the preovulatory LH and follicle stimulating hormone (FSH) surges (Jeffcoate et al., 1984; Haresign, 1985). In ovariectomised ewes treated with implants that constantly released progesterone and oestradiol, surges of both LH and FSH were observed when progesterone was withdrawn (Jeffcoate et al., 1984; Rawlings et al., 1984); the amplitude of these surges was on the low end of the normal range seen in intact ewes (Pant et al., 1977). In ovariectomised ewes, LH surges did not follow progesterone withdrawal, with serum oestradiol concentrations analogous to the luteal phase of an oestrous cycle, but if oestradiol was given after progesterone withdrawal to mimic levels seen in the follicular phase, surges resulted (Goodman et al., 1981; Fabre-Nys and Martin, 1991 ). The timing and amplitude of the LH surge appeared to be dependent on the dose and timing of administration of oestradiol after progesterone withdrawal; FSH was not monitored (Fabre-Nys and Martin, 1991; Goodman et al., 1981 ). The rate of increase of serum concentrations of oestradiol appeared to affect the timing and amplitude of the surge release of LH in ovariectomised ewes that had not been primed with progesterone (Rozell and Keisler, 1990). The purpose of the present study was to clarify the effect of dose and pattern of administration of oestradiol on the genesis of the surge release of both LH and FSH in ovariectomised ewes following progesterone withdrawal.
LB.J.K. Joseph et al. / Animal Reproduction Science 34 (1994) 217-230
219
Materials and methods
Experimental animals Sixty sexually mature crossbred ewes were kept outside in dry lots. During experiments the ewes were housed indoors under fluorescent lighting, with artificial daylength set to simulate natural daylength. The ewes were fed alfalfa pellets and alfalfa-brome hay, with cobalt-iodised salt and water freely available. Ewes were ovariectomised early in the breeding season and rested for at least 3-4 weeks before being used in this study.
Implants and infusions The steroid implants were prepared by mixing 10% (w/w) steroid with liquid silicone elastomer (RTV Silicone Elastomer, Factor II, Lakeside, AZ) and a curing catalyst. The oestradiol-17fl elastomer mixture was injected into silastic tubing of 0.32 cm inner diameter (i.d.) and 0.48 cm outer diameter (o.d.) (Dow Coming, Midland, MI ). When cured, the implants were cut into 0.5 or 6.0 cm lengths. The progesterone elastomer mixture was injected into tygon tubing moulds (Norton Plastics, Okbon, OH) of 0.48 cm i.d. The implants were removed from the moulds after 24 h and cut into 10 cm lengths. The implants were incubated in sterile 0.9% (w/v) saline for 24 h, at 37 °C, prior to insertion. Implants were introduced subcutaneously in the axillary region under local anaesthesia (Duracaine, Rogar/STB Inc., Montreal, Que., Canada). The release pattern of steroid from the implants has been described previously (Jeffcoate et al., 1984; Rawlings et al., 1984; Joseph et al., 1992). For infusions and collection of blood, ewes were catheterised in both jugular veins with vinyl tubing ( 1.00 mm i.d., 1.50 mm o.d.; SV-70, Dural Plastics and Engineering, Dural, N.S.W., Australia). Infusions were carded out using a ten-channel peristaltic pump (Manostat, New York), connected to the right jugular vein via silicone rubber tubing (2.38 mm i.d., 3.97 mm o.d. ).
Experimental protocol All ewes received a progesterone implant on Days 0, 2, 4, 6 and 10 of the study. Two, one and two progesterone implants were removed at 09: 00 h and 21:00 h on Day 11 and 09:00 h on Day 12, respectively (Fig. 1 ). Oestradiol implants were also inserted on Day 0 of the study. Forty-eight ewes received an oestradiol implant which was 0.5 cm in length, and 12 ewes received an implant 6 cm in length (Table 1 ). The oestradiol implants were left in place for the duration of the experiment. When progesterone implants were removed, additional treatments with oestradiol were imposed; these are summarised in Table 1 and Fig. 2. Twenty-four and six ewes treated respectively
220
I.B.J.K. Joseph et al. / Animal Reproduction Science 34 (1994) 217-230
6.0-
5.0-
4.0-
3.o 2.0
1.0
0.0
-
-2
|
0
.
1
2
•
i
4
•
i
.
6
i
8
•
i
10
-
i
12
•
=
-
14
Day Fig. 1. The temporal pattern of serum concentrations of progesterone (.(+ SEM) in long-term ovariectomised ewes given a 10 cm × 0.48 cm solid silastic rod containing progesterone on Days 0, 2, 4, 6 and 10 (J,). Two, one and two progesterone implants were removed at 09:00 h and 21 : 00 h on Day 11 and 09: 00 h on Day 12, respectively (T).
with 0.5 cm and 6 cm oestradiol implants were infused with oestradiol in saline for 40 h from 22:00 h on Day 11 (1 h after the second removal of progesterone implants) to 14: 00 h on Day 13. In 12 ewes treated with 0.5 cm oestradiol implants, the strength of infusate was doubled every 5 h, starting from 0.01/zg h - 1, to a final dose of 1.5 #g h - ~given in the last 5 h (low dose oestradiol infusion, Table 1 ). In all other ewes infused with oestradiol, the strength of infusate was doubled every 5 h, starting form 0.02/zg h - ~and with 3.0/zg h -~ infused in the last 5 h (high dose oestradiol infusion, Table 1 ). Two groups of six ewes treated with either 0.5 or 6 cm oestradiol implants, were infused with saline for 40 h from 22: 00 h on Day 11 to 14: 00 h on Day 13 (control, Table 1 ). In one group of ewes treated with 0.5 cm oestradiol implants, 10/zg of oestradiol in oil was injected (i.m.) 24 h after the last progesterone implant was removed (09:00 h on Day 13, injection; see Table 1 and Fig. 2). Finally, in one group of ewes treated with 0.5 cm oestradiol implants, one additional 0.5 cm oestradiol implant was introduced 12 h after the last progesterone implant was removed (21:00 h on Day 12, implant; see Table 1 and Fig. 2 ).
Blood sampling and analysis Daily blood samples were taken throughout the study for estimation of progestcrone concentration. Hourly samples were collected from 10: 00 h on Day 11 ( 1 h after onset of removal of progesterone implants) to 22: 00 h on Day
I.B.J.K. Joseph et al. /Animal Reproduction Science 34 (1994) 217-230
"0
0
0
~
. . . . .
ox.~
--i ~.
.o~ "= d
"~
~.~ 0
~.~ r~l
~-~
0
~
+l+l+l+l+l+l
0
+1'~
o ~ ,~ ~ .-. ~
e
~ +1+1+1+1+1+1
!e: N ~ ua'~ +~
+I+I+I+I+I+I
,S
~
c5 V ¢.)
+1+1+1+1+1+1
~e ~N
•~
o
o
0
.o
Oo~-~
e-t ~ N e~ ~ 0
~.~
o ~
0.~
0
~
~.~
o
221
401
222
I.B.J.K. Joseph et al. / Animal Reproduction Science 34 (1994) 217-230
3o
"6
~
20
L~111 •
10 •
•
0
4O
73o
i
•
•
•
i
24
•
•
•
|
48
•
"
"
0
72
•
•
•
0
i
•
•
•
24
|
•
•
•
I
48
"
'
'
72
40-
C
V
3O
-6 20
lO
•
•
•
i 24
•
•
•
i
•
•
•
48
i
•
•
•
0
•
.
.
i
•
.
•
•
•
i
•
24
Time after
•
•
|
48
•
•
.
i 48
°1o°l 0
•
24
72
i
•
•
.
.
•
i 72
.
.
•
i
96
•
72
Progesterone (h)
Fig. 2. The temporal pattern of serum concentrations of oestradiol ( X + SEM) in long-term ovariectomised ewes treated with oestradiol and progesterone releasing implants. Oestradiol implants 0.5 cm×0.48 cm ( a ) - ( d ) , or 6.0 cm×0.48 cm (e) were inserted on Day 0 and progesterone implants on Days 0, 2, 4, 6 and 10. Two, one and two progesterone implants were removed at 09: 00 h and 21 : 00 h on Day 11 and 09: 00 h on Day 12, respectively• Serum oestradiol concentrations are shown for 72 h following the first removal of progesterone implants (09:00 h on Day 11 to 09:00 h on Day 14). Over this period of time, additional oestradiol treatments were given as follows: (a) low dose or (b), (e) high dose infusion from 22:00 h on Day 11 until 14:00 h on Day 13 (0.5 cm implant+low dose infusion (F--t); 0.5 cm implant+high dose infusion (~- -1); 6.0 cm implant+high dose infusion (~- -t); (c) injection ( i.m. ) at 09: 00 h on Day 13 (0.5 cm implant + injection ( ~ ) ) or (d) an extra 0.5 cm × 0.48 cm implant at 21 : 00 h on Day 12 (0.5 cm implant + 0.5 cm implant ( ~ ) ). Further details of oestradiol doses are given in the text•
15, to characterise the surge release of gonadotrophins. Every second sample was analysed for concentrations of oestradiol. On Days 8 and 12, blood samples were taken every 10 min for 6 h to characterise pulsatile LH secretion.
L B.J.K. Joseph et al. /Animal Reproduction Science 34 (1994) 217-230
223
The sampling on Day 12 started 1 h after the last progesterone implant was removed.
Hormone assays Serum concentrations of FSH, LH, progesterone and oestradiol were determined in validated radioimmunoassays (Currie and Rawlings, 1989; Joseph et al., 1992). Concentrations of FSH and LH are expressed in terms of NIDDK-oFSH-RP 1 and NIDDK-oLH-24, respectively. The sensitivity of the gonadotrophin and steroid assays is expressed as the lowest standard different from zero, using an unpaired t-test. The limits of detection were 0.25 ng m l - 1 and 0.06 ng m l - ~ for FSH and LH respectively. Intra- and inter-assay coefficients of variation (CVs) for ovine reference sera with mean FSH concentrations of 14.1 ng m l - 1 or 1.13 ng m1-1 were 6.6% ( n = 8 ) and 9.6% ( n = l l ) or 7.6% ( n = l l ) and 9.7% (n = 110), respectively. Intra- and inter-assay CVs for ovine reference sera with a mean LH concentration of 3.01 ng ml -~ or 0.15 ng m1-1 were 5.0% ( n = 18) and 9.9% ( n = 110) or 9.6% ( n = 18) and 14.9% ( n = 110), respectively. The limits of detection for the oestradiol and progesterone assays were 1 pg ml-1 and 50 pg ml-1, respectively. For sera with oestradiol concentrations of 23 pg m1-1, 11.3 pg ml -~ or 8.8 pg ml -~, intra- and inter-assay CVs were 7.3% ( n = 2 ) and 9.1% ( n = 1 0 ) , 11% ( n = 2 ) and 11.6% ( n = 1 0 ) or 9.4% ( n = 2 ) and 12.5% ( n = 11 ), respectively. For sera with progesterone concentrations of 1.07 ng ml-~ or 2.15 ng ml-1, intra- and inter-assay CVs were 9.1% ( n = 7 ) and 11.4% ( n = 4 2 ) or 6.8% ( n = 7 ) and 9.5% ( n = 4 0 ) , respectively.
Statistical analysis The PULSAR program (Merriam and Wachter, 1982) was used to identify LH pulse frequency (pulses h - 1), amplitude (ng m l - ~), basal serum LH and mean serum LH concentrations (ng m l - 1 serum ) in blood samples taken every 10 min for 6 h on Days 8 and 12 (basal serum LH values are those after removal of pulses). The values of the G parameters used were 4.40, 2.60, 1.92, 1.46 and 1.13 for parameters 1, 2, 3, 4 and 5, respectively. The values assigned to the Baxter parameters were 0, 0.39 and 1.71 for A, B and C, respectively. According to this method, serum FSH concentrations did not show evidence of pulsatility. The surges of LH and FSH were identified visually. Data are presented as means and standard errors of the mean. Data were analysed by one-way and two-way analysis of variance as appropriate. Repeated measures analysis of variance was employed for the hormonal data resulting from blood samples collected every 10 min for 6 h on Days 8 and
224
LB.J.K. Joseph et al. /Animal Reproduction Science 34 (1994.) 217-230
12. After analysis of variance, individual treatment effects were examined by test of least significant differences.
Results
Serum steroids concentration: temporal patterns The temporal pattern of serum concentrations of progesterone produced by the progesterone implant regimen are shown in Fig. 1. The peak concentrations of oestradiol produced by the various treatments applied after progesterone withdrawal are shown in Table 1. The patterns of oestradiol produced by the various treatments are shown in Fig. 2. Serum progesterone concentration rose up to Day 8, and thereafter declined as implants were removed on Days 11 and 12. Peak progesterone concentrations varied quite markedly amongst ewes, as did the timing of the peak (Fig. 1 ). Serum concentrations of oestradiol produced by the two sizes of implants (0.5 cm × 0.48 cm vs. 6.0 cm × 0.48 cm) differed significantly ( P < 0.05 ) (Table 1 ). Infusion of oestradiol at either low or high dose produced peak levels of oestradiol that differed and were greater than those in control, non-infused ewes ( P < 0.05). Peak oestradiol concentration in ewes with small or large oestradiol implants (0.5 cm vs. 6 cm implants) did not differ when infused with the high dose of oestradiol ( P > 0.05 ). The peak concentration of oestradiol created by either injecting oestradiol or adding an extra 0.5 cm implant, after progesterone withdrawal, to ewes with the 0.5 cm implant were similar to ewes with a 0.5 cm implant and receiving the low dose infusion or ewes with the 6 cm oestradiol implant alone (Table 1 ). Following progesterone withdrawal, serum oestradiol concentrations in ewes treated with only one oestradiol implant were fairly constant. Infusion of oestradiol produced a gradual rise in oestradiol concentrations over the period of infusion (Fig. 2 ). Injection of oestradiol or insertion of a second oestradiol implant, produced a short-lived peak in concentration of oestradiol (less than 3 h), followed by a rapid decline. Following the peak, oestradiol levels were maintained at a higher level in ewes with two 0.5 cm oestradiol implants compared with those with only one (Fig. 2).
Gonadotrophin surges No gonadotrophin surges were seen following progesterone withdrawal in the presence of one 0.5 cm oestradiol implant (Table 1 ). In the other groups of ewes treated with 0.5 cm implants, gonadotrophin surges were seen in 11 / 12, 3/6 and 4/6 of the ewes receiving the high dose infusion, injection or added implant; in the low dose infusion group, 6/12 ewes had LH surges but only 1/12 had an FSH surge. In ewes treated with 6 cm implants, 5/6 had
LB.J.K. Joseph et al. / Animal Reproduction Science 34 (1994) 217-230
225
Table 2 Mean and basal serum concentrations o f L H and LH pulse amplitude and frequency in long-term ovariectomised ewes treated with progesterone releasing implants for 12 days and fitted with 0.5 c m × 0 . 4 8 cm or 6.0 c m × 0 . 4 8 cm oestradiol implants. Blood samples were taken every 10 min for 6 h on Days 8 and 12 o f treatment. The last progesterone implant was removed 1 h before sampling on Day 12. With progressterone withdrawal, three groups of ewes received either low or high dose infusions of oestradiol starting at 22:00 h on Day 11. See text and Fig. 1 for details of treatments Length of oestradiol implant (cm)
Post-progesterone Serum LH concentration oestradiol (ng ml- 1) treatment Mean Basal
LH pulse
Frequency (pulse h -1 )
Amplitude (ngm1-1 )
Day 8 0.5 6.0
0.35+0.07 b 0.17+0.01 ~
0.17+0.03 ~ 0.10+0.00 ~
0.56+0.11 a 0.61 +0.08 ~
0.84+0.22 b 0.42+0.06 ~
0 . 7 4 + 0 . 1 0 °a 0 . 7 8 + 0 . 1 5 *~b 1.31 + 0 . 2 6 *b 1.06 + 0.02 *b 0.92 + 0.02 *a
0 . 4 9 + 0 . 0 6 *ab 0 . 4 6 + 0 . 0 9 *ab 1.00+0.21 °c 0 . 5 4 + 0.02 °b 0.40 + 0.03 °~
1.23+0.12 *ab 0 . 9 7 + 0 . 1 4 *~ 1.35+0.13 *b 1.51 + 0 . 0 6 *b 1.36 + 0.07 *b
0.63+0.09 a 0 . 8 4 + 0 . 1 7 ab 0.79+0.09 a 1.12+0.05 °b~ 1.20 + 0.07 *c
Day 12 0.5 0.5 0.5 6.0 6.0
Control Low dose High dose Control High dose
*Significant differences between Day 8 and Day 12 ( P < 0.05).
Different superscripts within columns indicate signifcant differences between treatments for Day 8 and Day 12 separately ( P < 0.05 ).
gonadotrophin surges when no other oestradiol treatment was given and 6/6 had gonadotrophin surges when a high dose infusion was given. LH surges of similar amplitude followed progesterone withdrawal in ewes with 0.5 cm oestradiol implants and receiving the low dose infusion, injection or extra oestradiol implant and in ewes with one 6 cm oestradiol implant alone or with additional high dose infusion (Table 1 ). The ewes treated with a single 0.5 cm oestradiol implant and high dose infusion had LH surges of the greatest amplitude (Table 1 ) ( P < 0 . 0 5 ) . Latency from progesterone withdrawal (removal of first implant) to the peak of the LH surge was shorter in ewes receiving the high dose oestradiol infusion compared with all other ewes (Table 1 ) ( P < 0 . 0 5 ) . Unlike LH, the amplitudes of FSH surges were lower in all ewes with 6 cm implants than in those with 0.5 cm implants, with the exception of the one ewe in the low dose infusion group that had a surge. High dose infusion resulted in a mean FSH surge amplitude of 19.5_ 1.5 ng m l - l in ewes with 0.5 cm oestradiol implants, compared with 8.7 _+0.0 ng ml-~ in the one ewe having an FSH surge in the low dose infusion group. Again, unlike LH, the high dose infusion of oestradiol did not significantly reduce the latency from progesterone withdrawal to the FSH surge peak (Table 1 )
(P>0.05).
226
t.B.J.K. Joseph et al. /Animal Reproduction Science 34 (1994) 217-230
Pulsatile secretion of LH Pulsatile secretion of LH was examined on Days 8 and 12 of the experiment (Table 2). During progesterone and oestradiol treatment on Day 8, mean serum concentrations of LH and LH pulse amplitude were lower in ewes with 6 cm oestradiol implants than in those with 0.5 cm implants. Following progesterone withdrawal, mean and basal serum concentrations of LH and LH pulse frequency increased but LH pulse amplitude only increased in ewes treated with the larger (6 cm) oestradiol implant. On Day 12 (after progesterone withdrawal), mean and basal serum concentrations of LH tended to be higher in ewes with 6 cm oestradiol implants alone or 0.5 cm implants and the high dose oestradiol infusion; LH pulse frequency was lowest in ewes with 0.5 cm implants and low dose oestradiol infusion.
Discussion The progesterone concentrations resulting from implant treatments in this study were very similar to those seen in normal oestrous cycles in the ewe, both in pattern and absolute values (Rawlings et al., 1977). The basal serum oestradiol concentrations resulting from one small implant (0.5 cm) in our results were similar to concentrations we have seen in the luteal phase of normal cyclic ewes (4.8 +_0.1 pg ml -~ ) (Rawlings and Cook, 1992). The peak oestradiol concentrations resulting from two small implants, one large implant (6 cm ), low dose infusion of oestradiol or injection of oestradiol, were in the upper end of the range for the follicular phase of normal cyclic ewes ( 12.7 _+3.3 pg ml- ~) (Rawlings and Cook, 1992 ). Oestradiol concentrations resulting from high dose infusions were almost twice peak follicular phase levels. In previous studies with progesterone/oestradiol treated ovaricctomiscd ewes, basal concentrations of oestradiol like those of the luteal phase of cyclic ewes, did not result in LH surges following progesterone withdrawal (Goodman et al., 1981 ). However, if additional oestradiol was given after progesterone withdrawal to raise serum concentrations of oestradiol to those in the follicular phase of the oestrous cycle, LH surges were seen (Goodman et al., 1981; Fabrc-Nys and Martin, 1991 ). In addition, if serum concentrations of oestradiol were high but constant in oestradiol/progesterone treated ovaricctomised ewes, LH and FSH surges resulted from the withdrawal of progesterone (Jeffcoate et al., 1984; Rawlings et al., 1984). In our present work, progesterone withdrawal against constant basal serum concentrations of oestradiol, as expected (6.6_+ 1.2 pg ml-~; Table 1 ), did not result in gonadotrophin surges. LH and FSH surges were seen with all oestradiol treatments that resulted in peak serum concentrations of oestradiol that were within or greater than the range of values for the follicular phase of the oestrous cycle
LB.J.K. Joseph et aL / Animal Reproduction Science 34 (1994) 217-230
227
of the ewe ( 15-19 pg m l - ~; Table 1 ), regardless of whether oestradiol levels were constant or rising. LH surge amplitudes did not differ amongst these treatment groups, but not all ewes responded. This would suggest that these oestradiol concentrations were around threshold for the induction of gonadotrophin surges. Therefore, it would appear that an absolute level of oestradiol is required to cause a gonadotrophin surge following progesterone withdrawal, but that a particular pattern of increase is not important. In the present study, it appeared that LH surge amplitude was low in response to two treatments, the low dose oestradiol infusion into ewes with 0.5 cm oestradiol implants and ewes fitted with 6.0 cm oestradiol implants alone, even though follicular phase oestradiol concentrations were seen. These apparent differences were not significant for LH but they were for FSH; in fact, for FSH the former treatment resulted in a surge in only one ewe. It is difficult to explain why the low dose oestradiol infusion was ineffective in causing FSH surges. The 6.0 cm oestradiol implant alone resulted in FSH and LH surges in a fairly consistent manner but the surge amplitudes were low. Low surge amplitudes have been noted previously following progesterone withdrawal in oestradiol/progesterone treated ovariectomised ewes, when serum oestradiol concentrations were high but constant (Jeffcoate et al., 1984; Rawlings et al., 1984 ). Clearly, progesterone withdrawal and some degree of increase in serum concentrations of oestradiol are required to optimally induce the preovulatory gonadotrophin surge. In the present experiment, when the high dose infusion of oestradiol was given following progesterone withdrawal, LH and FSH surges were consistently produced. In addition, in ewes with 0.5 cm oestradiol implants and high dose oestradiol infusion, LH surge amplitude was higher than all other treatments and in fact, higher than in most intact ewes (Rawlings and Cook, 1992). It was of interest that the high peak serum oestradiol concentration produced by the high dose infusion in ewes with 0.5 cm oestradiol implants, in our work, did not cause extremely high FSH surge amplitudes as it did for LH. There is certainly an indication in our data that the FSH surge is less responsive to the oestradiol positive feedback process. The high dose infusion given to ewes with the large 6.0 cm oestradiol implant depressed LH and particularly FSH surge amplitudes. This apparent dose dependency for induction of LH surges and amplitude of the LH surge, with a depression at the highest serum oestradiol concentrations, has been indicated previously (Goodman et al., 1981; Rawlings et al., 1984). In general, in our work, the higher the peak serum concentration of oestradiol produced in ovariectomised ewes after progesterone withdrawal, the shorter the latency to the LH surge; this was not as clear for FSH. This effect has been seen previously for LH (Karsch et al., 1980; Goodman et al., 1981; Fabre-Nys and Martin, 1991 ). It has also been previously suggested that, in ovariectomised ewes with no progesterone priming, the rate of increase in serum concentrations of oestradiol influences the timing of the LH surge
228
LB.J.K. Joseph et al. /Animal Reproduction Science 34 (1994) 217-230
(Rozell and Keisler, 1990). We saw little evidence for this in our study. FabreNys and Martin ( 1991 ) have also shown that the timing of the increase in serum levels of oestradiol after progesterone withdrawal influences LH surge amplitude and timing in progesterone-primed ovariectomised ewes. The serum concentrations of oestradiol required to consistently elicit gonadotrophin surges in our work tended to be high compared with peak serum concentrations of oestradiol in intact ewes (Rawlings and Cook, 1992). However, the animals used in the present study were long-term ovariectomised ewes. It has been shown that negative feedback suppression ofgonadotrophin secretion by both progesterone and oestradiol decreases with time in the ovariectomised ewe (Karsch et al., 1977; Joseph et al., 1992); it is possible, therefore, that positive feedback sensitivity to physiological levels of oestradiol may also change with time. It is of interest that the basal or lowest serum oestradiol concentrations observed in the present experiment were similar to those that appeared to be at a threshold for the negative feedback regulation of LH secretion during the breeding season in long-term ovariectomised ewes (Joseph et al., 1992). In the present work, these serum concentrations ofoestradiol did not cause gonadotrophin surges but some of the higher concentrations of oestradiol did, indicating a clear differentiation between the threshold for the negative and positive feedback effects of oestradiol on gonadotrophin secretion. Observations of pulsatile LH secretion on Days 8 and 12 produced results that were largely as expected (Goodman and Karsch, 1980; Rawlings et al., 1984; Joseph and Rawlings, 1990). Progesterone largely suppresses LH pulse frequency and oestradiol LH pulse amplitude (Goodman and Karsch, 1980). On Day 8 the larger oestradiol implant resulted in a greater suppression of LH pulse amplitude and mean serum concentration of LH than the small implant (Table 2). As expected, LH pulse frequency rose by Day 12 (after progesterone removal) (Karsch et al., 1983 ). Interestingly, basal serum concentrations of LH also rose and together with the frequency effect this resulted in an increase in mean serum LH concentrations. On Day 12 there was some tendency toward variation between treatments in parameters of pulsatile LH secretion, but this was not consistent (Table 2). This could have been because the oestradiol infusions had only been running for 11 h and were still at fairly low doses by the time the intensive blood sampling was done. The increased LH pulse amplitude on Day 12 in ewes with 6.0 cm oestradiol implants was not expected and is difficult to explain. In conclusion, following progesterone removal in long-term ovariectomised ewes treated with oestradiol and progesterone releasing implants, gonadotrophin surges resulted in some but not all ewes when serum concentrations of oestradiol were in the range seen in intact ewes in the follicular phase of an oestrous cycle. The pattern of increase in oestradiol did not appear critical for the initiation of a gonadotrophin surge. Although constant high levels
LB.J.K. Joseph et al. / AnimaI Reproduction Science 34 (1994) 217-230
229
of oestradiol consistently elicited gonadotrophin surges they did not give surges of as great an amplitude as those arising from rising serum concentrations of oestradiol. Increasing serum concentrations of oestradiol above follicular phase levels consistently induced gonadotrophin surges, increased LH but not FSH surge amplitude and decreased latency to the LH surge. The highest peak serum concentrations of oestradiol used in this study actually depressed LH and FSH surge amplitude. The mechanism leading to the FSH surge may be somewhat less responsive to oestradiol than that leading to the LH surge. Acknowledgements The authors thank M. Buckley and her staff for animal care. Gonadotrophin assay reagents were obtained from NIDDK. I.B.J.K. Joseph was a recipient of a Canadian Commonwealth Scholarship. This work was supported by NSERC, Canada.
References Caraty, A., Locatelli, A. and Martin, G.B., 1989. Biphasic response in the secretion of gonadotrophin-releasing hormone in ovariectomised ewes injected with oestradiol. J. Endocrinol., 123: 375-382. Clarke, I.J. and Cummins, J.T., 1985. Increased gonadotropin-releasing hormone pulse frequency associated with estrogen-induced luteinizing hormone surges in ovariectomised ewes. Endocrinology, 116: 2376-2383. Currie, W.D. and Rawlings, N.C., 1989. Fluctuations in responsiveness of LH and lack of responsiveness of FSH to prolonged infusion of morphine and naloxone in the ewe. J. Reprod. Fertil., 86: 359-366. Emons, G., Schuppe, H., Peter, M., Brack, C. and Ball, P., 1986. Modulation of LH secretion in ovariectomized ewes by constant infusions of estradiol and 4-hydroxyoestradiol--effects of varying infusion time and of high estrogen doses. Acta Endocrinol., 113:219-225. Fabre-Nys, C. and Martin, G.B., 1991. Roles of progesterone and oestradiol in determining the temporal sequence and quantitative expression of sexual receptivity and the preovulatory LH surge in the ewe. J. Endocrinol., 130: 367-379. Goding, J.R., Cart, K.J., Brown, J.M., Kaltenback, C.C., Cumming, I.A. and Mole, B.J., 1969. Radioimmunoassay for ovine luteinising hormone. Secretion of luteinizing hormone during estrus and following estrogen administration in the sheep. Endocrinology, 85:133-142. Goodman, R.L. and Karsch, F.J., 1980. Pulsatile secretion of luteinizing hormone; differential suppression by ovarian steroids. Endocrinology, 107:1286-1290. Goodman, R.L., Legan, S.J., Ryan, K.D., Foster, D.L. and Karsch, F.J., 1981. Importance of variation in behavioral and feedback action of oestradiol to the control of seasonal breeding in the ewe. J. Endocrinol., 89: 229-240. Haresign, W., 1985. Comparison of the rate of decline in plasma progesterone concentration at a natural and progesterone-synchronized oestrus and its effect on tonic LH secretion in the ewe. J. Reprod. Fertil., 75: 231-236. Hauger, R.L., Karsch, F.J. and Foster, D.L., 1977. A new concept for control of the oestrous cycle of the ewe. Based on the temporal relationships between luteinizing hormone, oestra-
2 30
LB.J.K. Joseph et al./Animal Reproduction Science 34 (1994) 217-230
diol and progesterone in peripheral serum and evidence that progesterone inhibits tonic LH secretion. Endocrinology, 101: 807-818. Jeffcoate, I.A., Rawlings, N.C. and Rieger, D., 1984. Progesterone and the surge release of gonadotropins in the ewe. Domest. Anim. Endocrinol., 1:309-317. Joseph, I.B.J.K. and Rawlings, N.C., 1990. Infusion of estradiol during the luteal phase of the estrous cycle in the ewe completely eliminates LH pulses. Can. J. Anim. Sci., 70:1147-1150. Joseph, I.B.J.K., Currie, W.D. and Rawlings, N.C., 1992. Luteinising hormone and follicle stimulating hormone secretion in ovariectomised ewes: Effects of time post ovariectomy, season and oestradiol. J. Reprod. Fertil., 94:511-523. Karsch, F.J. and Foster, D.L., 1975. Sexual differentiation of the mechanism controlling the preovulatory discharge of luteinising hormone in sheep. Endocrinology, 97: 373-382. Karsch, F.J., Legan, S.J., Hauger, R.L. and Foster, D.L., 1977. Negative feedback action of progesterone on tonic luteinising hormone secretion in the ewe: Dependence on the ovaries. Endocrinology, 101: 800-806. Karsch, F.J., Legan, S.J., Ryan, K.D. and Foster, D.L., 1980. Importance of estradiol and progesterone in regulating LH secretion and estrous behaviour during the sheep estrous cycle. Biol. Reprod., 23: 404-413. Karsch, F.J., Foster, D.L., Bittman, E.L. and Goodman, R.L., 1983. A role for estradiol in enhancing luteinising hormone pulse frequency during the follicular phase of the estrous cycle of sheep. Endocrinology, 113:1333-1359. Merriam, G.R. and Wachter, K.W., 1982. Algorithms for the study of episodic hormone secretion. Am. J. Physiol., 243:E310-E318. Moenter, S.M., Caraty, A. and Karsch, F.J., 1990. The estradiol-induced surge ofgonadotropinreleasing hormone in the ewe. Endocrinology, 127:1375-1384. Pant, H.C., Hopkinson, C.R.N. and Fitzpatrick, R.J., 1977. Concentrations of oestradiol, progesterone, luteinizing hormone and follicle-stimulating hormone in the jugular venous plasma of ewes during the oestrous cycle. J. Endocrinol., 773: 247-255. Radford, H.M., Wheatley, I.S. and Wallace, A.L.C., 1969. The effects of oestradiol benzoate and progesterone on secretion of luteinizing hormone in the ovariectomised ewe. J. Endocrinol., 44: 135-136. Rawlings, N.C. and Cook, S.J., 1992. LH secretion around the time of the preovulatory gonadotrophin surge in the ewe. Anim. Reprod. Sci., 30: 289-299. Rawlings, N.C., Kennedy, S.W., Chang, C.H., Hill, J.R. and Henricks, D.M., 1977. Onset of seasonal anestrus in the ewe. J. Anita. Sci., 44: 791-797. Rawlings, N.C., Kennedy, S.W. and Henricks, D.M., 1978. Effect of active immunisation of the cyclic ewe against oestradiol-17ft. J. Endocrinol., 76: I 1-19. Rawlings, N.C., Jeffcoate, I.A. and Rieger, D.L., 1984. The influence of oestradiol-17fl and progesterone on peripheral serum concentration of luteinizing hormone and follicle stimulating hormone in the ovariectomized ewe. Theriogenology, 22: 473-488. Rozell, T.G. and Keisler, D.H., 1990. Effects of oestradiol on LH, FSH and prolactin in ovariectomized ewes. J. Reprod. Fertil., 88: 645-653.