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Biology of Reproduction 59, 1062-1068 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Equine Chorionic Gonadotropin Regulates Luteal Steroidogenesis in Pregnant Mares1

P.F. Daels2,a, B.A. Albrechta, , and H.O. Mohammeda

a Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The onset of eCG secretion in pregnant mares coincides with an increase in luteal steroid production and a relative shift toward androgen and estrogen synthesis. However, a cause-effect relationship between eCG and the shift in luteal steroidogenesis has not been demonstrated. In this study, we have investigated the effect of eCG on steroid production by the corpus luteum (CL) during equine pregnancy. All mares were supplemented with 44 mg altrenogest (a progestogen) per day on Days 18–50. Increasing doses of eCG were administered on Days 26–28, before the onset of endogenous eCG secretion, to four mares with and four mares without a functional CL (prostaglandin F2{alpha} administered on Day 18). Four mares with a functional CL received no exogenous eCG. In eCG-treated mares without a functional CL, progestin, androstenedione, and estrogen concentrations did not significantly increase after exogenous eCG administration or endogenous eCG secretion. In eCG-treated mares with a functional CL, progestin and estrogen production increased significantly after exogenous eCG administration and endogenous eCG secretion, whereas androstenedione concentrations tended to increase following exogenous eCG and increased significantly following endogenous eCG secretion. In mares with a functional CL that did not receive exogenous eCG, progestin and estrogen concentrations increased and androstenedione concentrations tended to increase only after the onset of endogenous eCG secretion. These data demonstrate that the increase in luteal steroidogenesis that coincides with the onset of eCG secretion is induced by eCG and results in an increase in luteal androgen and estrogen synthesis. Our findings support the hypothesis that eCG has a luteotropic action in pregnant mares.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The endocrine dynamics in the pregnant mare are unique when compared to those in other species. During the first 3 mo of gestation, pregnancy is maintained by progesterone secreted by the maternal gonads. The primary corpus luteum (CL) or CL of conception is the main source of progestin for the first 40–50 days and continues to contribute until its demise around Day 120–150 of pregnancy [1–3]. Holtan et al. [4] reported that in addition to progesterone, 5{alpha}-pregnane-3,20-dione (5{alpha}-DHP) and 3ß-5{alpha}-pregnant-20-one (3ß-5P) are also detectable and that increases in these parallel that in progesterone during early pregnancy [4]. The authors further suggested that during the first weeks of pregnancy, 5{alpha}-DHP and 3ß-5P are likely secreted by the maternal CL and may be unrelated to pregnancy status. Similar increases in these progestins (5{alpha}-DHP and 3ß-5P) are also observed during the luteal phase of nonpregnant mares (D. Holtan, personal communication). Between Days 40 and 150 of gestation, secondary CL may develop and provide an additional source of progestin [5–7]. However, secondary CL do not develop in all pregnant mares, and their development in time and numbers is extremely variable, suggesting that these may not be essential to the outcome of pregnancy. After Days 120–160, primary and secondary CL cease to function, and from this point forward pregnancy is maintained by the secretion of progestins from the fetoplacental unit. It is assumed that luteal function is supported primarily by maternal pituitary LH until 35–40 days of pregnancy [8]. In nonpregnant mares, luteal LH receptors have been identified [9], and their numbers have been reported to increase during diestrus [10]. Passive immunization against pituitary extract resulted in smaller CL and premature regression, suggesting a role for pituitary support of luteal function [11, 12]. Human CG administered to mares in early diestrus results in an increase in circulating progestin concentrations [13, 14]. Around Day 35, chorionic girdle cells differentiate into endometrial cup cells, secreting eCG from approximately Days 35 to 120 of pregnancy [15–17].

Several observations indicate that eCG is the main luteotropic agent during the period of endometrial cup function. Studies comparing the life span of the primary CL in hysterectomized and pregnant mares suggest that the functional life span of the CL is extended by the luteotropic action of eCG [18–20]. In hysterectomized mares, the primary CL regressed between Days 70 and 140, but it did not regress until after Day 140 in pregnant mares. A gradual increase in circulating progestin concentrations has been associated in two studies with the onset of eCG secretion [20, 21]. In both studies, the increase in progestin secretion was observed before the development of secondary CL, suggesting that the increase is the result of an eCG-induced stimulation of the primary CL. Similarly, progestin production by tissue slices from primary and secondary CL increases during incubation with eCG [6]. Furthermore, it has been reported, on the basis of ultrasonographic observations, that the primary CL increases in size after Day 35 [21]. The increase in progestin production and the increase in size have been referred to as the resurgence of the primary CL [8].

It has been demonstrated that the resurgence of the primary CL coincides with an increase in androgen and estrogen secretion by the primary CL [22–24]. The onset of eCG secretion coincides closely with the rapid increase in luteal estrogen secretion and, to a lesser extent, luteal androgen secretion. No increases in androgen or estrogen concentrations are observed in progestogen-treated, pregnant mares without a functional CL, suggesting that the increases in circulating androgen and estrogen concentrations are the result of changes in luteal steroidogenesis [23, 24]. The ability of equine luteal cells to produce androgens and estrogen has also been demonstrated in vitro. Incubation with progesterone of equine luteal microsomes from nonconceptual cycles results in the production of 17{alpha}-hydroxyprogesterone and estrogen [25–27]. Recently, the same group of researchers has reported immunohistochemical evidence for the presence of aromatase in the equine CL of diestrus [28]. We have recently compared in vitro progesterone and estrogen production by luteal cells recovered during diestrus (Days 7–10) and pregnancy before (Days 30–35) and after (Days 40–45) the onset of eCG secretion and have demonstrated that estrogen synthesis, but not progesterone synthesis, increases during pregnancy and is highest during the time of eCG secretion [29].

The objective of the present study was to demonstrate whether eCG stimulates luteal androgen and estrogen production during pregnancy in mares. Exogenous eCG was administered to pregnant mares with and without a functional CL prior to the onset of endogenous eCG secretion, and the resulting changes in steroid secretion were characterized.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Twelve light-breed, pregnant mares aged 6–14 yr were used in this study. Mares were maintained on pasture and supplemented with hay, minerals, and water ad libitum.

Experimental Design

Mares were randomly assigned to one of three groups (n = 4 per group). Mares in group C served as controls with a functional CL and did not receive exogenous eCG. Mares in group CL+eCG had a functional CL and received increasing doses of exogenous eCG on Days 26–28 of pregnancy. Mares in group noCL+eCG had no functional primary CL and received increasing doses of exogenous eCG on Days 26–28 of pregnancy. Time of ovulation, pregnancy status, and ovarian dynamics during pregnancy were monitored by ultrasonography per rectum daily during estrus and every other day during pregnancy. Blood samples were collected daily on Days 20–50 of pregnancy, and plasma was analyzed for progestin, androstenedione, estrogen, and eCG concentrations.

Treatments

All mares received altrenogest (44 mg/day, ReguMate; Hoechst-Roussel, Agri-Vet, Somerville, NJ) to maintain pregnancy in those mares without a functional CL and to expose the remaining mares (with functional CL) to the same experimental conditions [22, 23, 30]. Altrenogest was administered per os daily from Days 18 to 50. Mares in group noCL+eCG received a luteolytic dose of prostaglandin F2{alpha} (PGF2{alpha}; 10 mg, i.m., Lutalyse; Upjohn Co., Kalamazoo, MI) on Day 18 of pregnancy. Exogenous eCG (Folligon; Intervet, The Netherlands) was administered by i.m. injection on Day 26 (4 IU/kg), Day 27 (10 IU/kg), and Day 28 (16 IU/kg) of pregnancy to mares in groups CL+eCG and noCL+eCG. All treatments were administered between 0800 and 0900 h for the duration of the study.

Blood Samples and Endocrine Analyses

Blood samples were collected by jugular venipuncture into heparinized tubes. Plasma was stored at -20°C until assayed for progestin, androstenedione, estrogen, and eCG.

Progestins Progesterone and closely related progestin concentrations in ether-extracted samples were determined by enzyme immunoassay [31]. The cross-reactivity of the antiserum (R4861; Dr. Bill Lasley, University of California, Davis, CA) is 21.4% for 11-hydroxyprogesterone, 29.5% for 5{alpha}-pregnane-3,20-dione, and less than 0.5% for altrenogest and other steroids tested. The limit of sensitivity was 0.11 ng/ml (zero standard ± 95% confidence interval). The interassay and intraassay coefficients were 14% and 7%, respectively.

Androstenedione Androstenedione concentrations were determined in ether-extracted samples by RIA using an anti-androstenedione antibody (G4; Dr. D.T. Armstrong, University of Western Ontario, London, ON, Canada) and tritiated androst-4-ene-3,17-dione (DuPont-NEN, Boston, MA). Stock buffer consisted of 0.1 M sodium phosphate containing 0.9% NaCl and 0.1% sodium azide (PBS), pH 6.8. All assay dilutions were made in PBS containing 0.1% gelatin (PBSG). For the assay, 200 µl of plasma was extracted with 2 ml of anhydrous ethyl ether and reconstituted in 100 µl of PBSG. On Day 1, 100 µl of antibody diluted to 1:3330 and 100 µl of trace (13 000 cpm/100 µl) were added to 100 µl of standard (6.25–800 pg/100 µl) or sample and incubated for 18–24 h at 4°C. On Day 2, free and bound radioactivity were separated by adsorption of unbound radiolabeled antigen with 1 ml dextran-coated charcoal solution (0.25% Norit A, 0.025% dextran T-70 in PBS) and incubated for 15 min at 4°C. Tubes were centrifuged at 4°C for 15 min at 2580 relative centrifugal force. Supernatants were decanted into scintillation vials with 5 ml of single-phase scintillation cocktail (Ultima Gold XR; Packard Instrument Co., Meriden, CT) and counted for 2 min. Previously reported cross-reactivities for the antiserum are 5% for 5{alpha}-androstane-3,17-dione, 2% for testosterone, and less than 1% for all other steroids tested [32]. Serial dilution of plasma samples from pregnant mares (Days 64 and 74 of pregnancy) and from a stallion produced inhibition curves parallel to the standard curve (data not shown). The limit of sensitivity was 20.7 pg/ml (ether-extracted zero standard ± 95% confidence interval). Extraction efficiency averaged 85%. Results were not corrected for extraction efficiency. The average percentage recovery of known amounts of androstenedione added to extracted samples of equine plasma was 100.6%. The correlation coefficient between expected and observed values was 0.993 with regression equation of y = 0.916x - 3.87. The interassay and intraassay coefficients were 8% and 7%, respectively.

Estrogen Estrogen concentrations were determined using a direct RIA without extraction [22, 33]. Tritiated estrone-3-sulfate (Dupont-NEN) was used for tracer, and the antibody (R-583, Dr. Bill Lasley, University of California, Davis, CA) was produced in rabbits and was directed against estrone-3-glucuronide that had been conjugated to BSA. With the use of estrone-3-sulfate as the standard (100%), the antibody cross-reacts 200% with estrone, 100% with estradiol-17ß, 50% with equilin, 38% with estrone-3-glucuronide, 21% with estradiol-3-sulfate, 6.8% with estradiol-3-glucuronide, and less than 0.5% with all non-estrogenic steroids tested. For the assay, plasma samples were diluted 1:6 in Tris buffer, and 0.30 ml was assayed. The limit of sensitivity was 0.084 ng/ml. The interassay coefficients of variation were 24% at 15% binding and 21% at 73% binding, and the intraassay coefficient of variation was 9%.

Equine CG Plasma eCG concentrations were determined by heterologous, double-antibody RIA [34]. Highly purified equine LH (E263B) and CG (PM230GB) (Dr. H. Papkoff, University of California, Davis, CA) were used for iodination and standards, respectively. The first antibody was a mouse anti-bovine LH-ß (monoclonal antibody 518B7; Dr. J.F. Roser, University of California, Davis, CA). The limit of sensitivity of the assay was 3.5 ng/ml. The interassay and intraassay coefficients were 4% and 9%, respectively.

Statistical Analysis

Three 5-day periods were examined to determine whether eCG stimulates luteal steroidogenesis. Period 1 (Days 20–24 inclusive) represents basal luteal steroidogenesis. Period 2 (Days 26–30 inclusive) represents luteal steroidogenesis in response to exogenous eCG administration. Period 3 (5 days beginning with the day of onset of endogenous eCG secretion) represents luteal steroidogenesis in response to endogenous eCG secretion. To control for individual variation in basal steroid production between mares, analyses of treatment effect were based on the change in steroid secretion between periods for individual mares. Steroid secretion was determined by calculating the area under the curve (estimation of total steroid production; expressed in ng per 5 days/ml for each 5-day period. The changes in steroid secretion for each hormone were subjected to two-factor ANOVA with group and period as the factors. Significant changes in luteal steroid production between groups for a given period were determined by Fisher's least-significant difference. Significant changes in luteal steroid production between periods within a group were determined by Fisher's least-significant difference. Student's t-tests were used to determine whether a change in steroid production was significantly different from zero. The day of onset of endogenous eCG secretion was defined as the day of gestation that was followed by two consecutive (daily) 2-fold or greater increases in eCG concentrations with a continuous increase on subsequent days. The day of onset of eCG secretion was analyzed by one-factor ANOVA and Tukey's pair-wise comparison. Significance was considered at p <= 0.05. All values are expressed as mean ± SD.

In the noCL+eCG group, plasma androstenedione concentrations appeared to change toward the end of the observation period. An additional period of steroid secretion was analyzed for this hormone. The period (period 4) includes 5 days beginning with the ninth day after the onset of eCG secretion. A Student's t-test was utilized to determine whether the increase was significantly different from zero.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Equine CG

The overall mean day of onset of eCG secretion (see Materials and Methods) was Day 36 ± 1.6 of pregnancy and ranged from Day 37 to 39, 35 to 38, and 34 to 37 for groups C, CL+eCG, and noCL+eCG, respectively. The mean day of onset of endogenous eCG secretion was not significantly different between groups. In all eCG-treated mares, eCG immunoreactivity increased during eCG treatment. After the last eCG administration, eCG immunoreactivity decreased and returned to pretreatment levels before the onset of endogenous eCG secretion in all mares (Fig. 1).



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  		FIG. 1. Individual circulating eCG concentrations in mares in groups C, CL+eCG, and noCL+eCG. Concentrations after Day 32 are aligned according to the onset of eCG secretion (Day 35). Mares in group C served as controls and received altrenogest daily (mares: 88, squares; 115A, plus signs; 280, diamonds; 210, triangles). Mares in group CL+eCG received altrenogest daily and increasing doses of eCG on Days 26–28 (indicated by solid bar along x-axis; mares: 114, squares; 115, plus signs; 117, diamonds; 276, triangles). Mares in group noCL+eCG received daily altrenogest, PGF2{alpha} on Day 18, and increasing doses of eCG on Days 26–28 (indicated by solid bar along x-axis; mares: 41, squares; 46, plus signs; 274, diamonds; 421, triangles).

Progestins

Group, period, and the group-by-period interaction had significant effects on the changes in progestin secretion. Mean changes in progestin production are summarized in Table 1, and individual progestin profiles are shown in Figure 2. After the onset of eCG secretion, progestin production (change from period 1 to 3) significantly increased in groups C and CL+eCG and remained constant in group noCL+eCG. Exogenous eCG (change from period 1 to 2) tended to stimulate progestin production in group CL+eCG (p < 0.065), while progestin levels tended to decline in group C (p < 0.086) and declined in group noCL+eCG. Progestin levels in the noCL+eCG mares were significantly lower than in groups C and CL+eCG. Progestin concentrations remained less than or equal to 0.32 ng/ml until Day 50 in all mares in group noCL+eCG except one (mare 274). In mare 274, progestin concentrations were less than 0.3 ng/ml until Day 48 of pregnancy, indicating complete regression of the primary CL following PGF2{alpha} administration. This mare ovulated between examinations on Days 47 and 48, by ultrasonography, and progestin concentrations increased above 1 ng/ml on Day 49.


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  		TABLE 1. Mean change in steroid production.a



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  		FIG. 2. Individual circulating progestin concentrations in mares in groups C, CL+eCG, and noCL+eCG. See legend for Figure 1 for details.

Comparison between groups indicated that the changes in progestin production were similar regardless of exogenous eCG administration (change from period 1 to 2) in mares with a CL (groups C and CL+eCG). The increases induced by endogenous eCG (change from period 1 to 3) in groups C and CL+eCG were similar and were significantly greater than those observed in group noCL+eCG.

Androstenedione

Group and period but not the group-by-period interaction had significant effects on the changes in androstenedione production. Mean changes in androstenedione production are summarized in Table 1, and individual androstenedione profiles are shown in Figure 3. After the onset of endogenous eCG secretion (change from period 1 to 3), plasma androstenedione concentrations increased significantly in group CL+eCG and tended to increase in group C (p < 0.10). Androstenedione concentrations in group noCL+eCG remained constant during period 3 (day of onset of endogenous eCG secretion and subsequent 4 days) and tended to increase during period 4 (Day 9 after the onset of endogenous eCG secretion and subsequent 4 days; p < 0.061). Exogenous eCG did not stimulate androstenedione production in group CL+eCG. During the same period (change from period 1 to 2), androstenedione concentrations decreased in group noCL+eCG (p < 0.038) and tended to decline in group C (p < 0.15).



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  		FIG. 3. Individual circulating androstenedione concentrations in mares in groups C, CL+eCG, and noCL+eCG. See legend for Figure 1 for details.

Comparison between groups indicated that changes in androstenedione production were similar between the groups for all periods.

Estrogen

Group, period, and the group-by-period interaction had significant effects on the changes in estrogen production. Mean changes in estrogen production are summarized in Table 1, and individual profiles are shown in Figure 4. After the onset of endogenous eCG secretion, plasma estrogen concentrations were significantly increased in mares with CL (groups C and CL+eCG) and remained constant in mares without a CL (group noCL+eCG). Exogenous eCG stimulated estrogen production in group CL+eCG. Estrogen production remained constant in groups C and noCL+eCG during the same period (change from period 1 to 2).



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  		FIG. 4. Individual circulating estrogen concentrations in mares in groups C, CL+eCG, and noCL+eCG. For details see legend for Figure 1.

Comparison between groups indicated that changes in estrogen production in groups C and CL+eCG were similar after the onset of endogenous eCG secretion but that both differed significantly from changes in group noCL+eCG.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this experiment further demonstrate a role for eCG in the regulation of luteal progestin, androgen, and estrogen secretion during equine pregnancy. Administration of exogenous eCG on Days 26–28 of pregnancy resulted in a transient but significant increase in luteal progestin and estrogen secretion, while no change was observed in control mares with a functional CL for the same period. Similarly, an increase in progestin and estrogen secretion was observed in association with the onset of endogenous eCG secretion in all mares with a functional CL. Androstenedione secretion tended to increase in response to the eCG administrations whereas androstenedione secretion in control mares with a functional CL tended to decrease during the same period, suggesting that exogenous eCG might stimulate luteal androgen secretion as well. This is supported by the observation that significant increases in androstenedione secretion were observed in association with the onset of eCG secretion in all mares with a functional CL while no change was observed in mares without CL. The increases in progestin secretion associated with exogenous or endogenous eCG could not be attributed to the formation of secondary CL, since none of the mares developed a secondary CL during the periods that were compared. We have previously observed that altrenogest administration during pregnancy results in a delay and reduction in the development of secondary CL, with the first secondary CL occurring at a later stage of gestation and a decrease in the total number of secondary CL [22–24, 35]. We have also previously observed that altrenogest supplementation results in a decrease in basal estrogen levels in pregnant mares [23]. In the present study and in the previous study, the decrease in basal estrogen levels appeared to be due to depressed follicular activity. However, we have previously demonstrated that the relative increase in luteal estrogen secretion at the onset of eCG secretion was similar in altrenogest-treated and untreated pregnant mares [23].

Estrogen concentrations and to a lesser extent progestin and androstenedione decrease after the initial increase stimulated by exogenous eCG. This likely reflects the clearance of exogenous eCG from the circulation. Measurement of eCG immunoreactivity after exogenous eCG administration indicates that in all mares, eCG levels decrease to levels near or equal to pretreatment levels before the onset of endogenous eCG secretion. In contrast, the decrease in luteal estrogen concentrations that follows the initial increase in estrogen secretion at the onset of endogenous eCG secretion occurs while endogenous eCG secretion continues to increase. The lack of a continued increase in luteal estrogen production during the period of increasing eCG concentrations has previously been reported, but no mechanism for this apparent insensitivity of the CL to eCG has yet been proposed [22, 23, 30].

In mares without a functional CL, secretion of the three steroids measured decreased during the period of eCG administration and remained unchanged in response to endogenous eCG. The number and size of follicles were similar between control mares and eCG-treated mares without CL for equivalent periods (data not shown). These observations indicate that eCG does not induce detectable changes in follicular steroidogenesis and suggest that the CL is the primary target within the maternal ovary.

After the end of period 3, there was a tendency for plasma androstenedione and to a lesser extent estrogen concentrations to increase in mares without a functional CL. These increases in the absence of a CL are likely to be a reflection of changes in fetoplacental steroidogenesis as previously reported [23, 24]. Thus increases observed after period 3 in any of the three experimental groups do not represent long-term, eCG-induced changes in luteal steroidogenesis.

The dose of exogenous eCG chosen in the present study was based on our previous studies on eCG levels and was aimed at simulating the endogenous increase in bioactive eCG previously reported [36]. The rate of increase and the peak estrogen concentrations induced by exogenous eCG administration and endogenous eCG secretion in eCG-treated mares with CL appeared similar. This suggests that the dosage of eCG used in the present study closely paralleled the onset of endogenous eCG secretion and resulted in a response that was within the physiological range observed at the onset of endogenous eCG secretion. After eCG administration (eCG-treated mares with CL) and after the onset of eCG secretion (all mares with CL), changes in estrogen production were relatively more pronounced compared to those in progestin and androstenedione. The exact function of the eCG-induced shift in luteal steroidogenesis remains unclear. It is possible that the increase in androgen concentrations in the peripheral circulation is merely a reflection of increased substrate production for aromatization, and therefore, circulating concentrations may not accurately reflect changes in synthesis. We have recently demonstrated that in the equine CL, steady-state levels of mRNA for P450 17{alpha}-hydroxylase/17,20-lyase remain basal until after the onset of eCG secretion [37, 38]. In contrast, mRNA levels for cytochrome P450 aromatase are higher in pregnant mares before the onset of eCG secretion and decrease significantly after the onset of eCG secretion. These observations suggest that perhaps 17{alpha}-hydroxylase/17,20-lyase activity is the rate-limiting step in luteal estrogen synthesis and that the eCG-induced increase in estrogen synthesis is primarily the result of substrate availability [39]. The mechanism(s) involved in eCG-regulated luteal estrogen synthesis also remains unclear. At the onset of eCG secretion, a rapid increase in estrogen concentrations is followed by a slight decrease and fairly constant levels despite a continuous increase in circulating eCG concentrations. There is no information available in equids to suggest that this is the result of down-regulation of eCG receptors or changes in postreceptor events. Steady-state levels of mRNA and protein for P450 aromatase are significantly lower around Day 45 of gestation than during diestrus or pregnancy before the onset of eCG secretion [39, 40], suggesting that expression of P450 aromatase may be down-regulated. However, no data are currently available as to the level of P450 aromatase activity within the equine CL.

Observations in other species lead us to speculate that in addition to the direct stimulatory effect on luteal estrogen and progestin secretion, eCG may also have an indirect stimulatory effect on progestin secretion that is mediated by estrogen. In species such as the pig, human, and rat, it has been demonstrated that luteal estrogen acts as a luteotropin and directly stimulates luteal progestin synthesis [41–45]. In pregnant rats, the luteotropic action on the CL is due to LH stimulation of estradiol, and it is estrogen synthesized within the CL that, together with decidual luteotropin, sustains progestin production (reviewed in [45]). In nonpregnant pigs and human and nonhuman primates it has been demonstrated that oxytocin stimulates estrogen synthesis and that estrogen stimulates progestin synthesis [41, 42, 46]. The experimental data on the in vitro effect of eCG on luteal steroidogenesis in pregnant mares are unclear and incomplete. Squires et al. [6] reported that addition of eCG to the incubation medium increased progestin production by slices of primary and secondary CL from pregnant mares; however, estrogen production was not determined. Some other investigators have reported an increase in progestin production when equine luteal cells from diestrus or pregnancy are incubated with LH, hCG, or eCG, whereas still others did not observe an effect of LH or hCG on luteal progestin production in vitro [13, 29, 47–49]. Further studies are needed to determine whether the luteotropic effect of eCG is primarily a direct effect on luteal progestin production or whether it is mediated by estrogen as has been demonstrated in other animal models.

In conclusion, the results of this study further demonstrate that eCG regulates luteal steroidogenesis in pregnant mares and induces a pronounced shift toward luteal estrogen secretion. The eCG-induced changes in luteal steroid production at the onset of eCG secretion, combined with the temporal association between luteal regression and end of eCG secretion later in pregnancy, support the hypothesis that eCG has a luteotropic role during pregnancy.


   ACKNOWLEDGMENTS
 
Special thanks to Betty Hansen for endocrine analyses; Dr. J. Roser, Dr. H. Papkoff, Dr. B. Lasley, C. Munro, and Dr. T. Armstrong for generously providing the antibodies and purified proteins used in the analyses; Hoechst-Roussel, Agri-Vet for generously donating ReguMate; and Carol Collyer and staff at the Equine Research Park for animal care.


   FOOTNOTES
 
1 This work was supported by the Harry M. Zweig Memorial Fund for Equine Research at Cornell University. Back

2 Correspondence: Peter Daels, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. FAX: 607 253 3055; pfd1{at}cornell.edu Back

Accepted: June 19, 1998.

Received: October 20, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sharp DC, McDowell KJ, Weithenauer J, Thatcher WW. The continuum of events leading to maternal recognition of pregnancy in mares. J Reprod Fertil Suppl 1989; 37:101–107.[Medline]
  2. McDowell KJ, Sharp DC, Grubaugh W, Thatcher WW, Wilcox CJ. Restricted conceptus mobility results in failure of pregnancy maintenance in mares. Biol Reprod 1988; 39:340–348.[Abstract]
  3. McDowell KJ, Sharp DC, Peck LS, Cheves LL. Effect of restricted conceptus mobility on maternal recognition of pregnancy in mares. Equine Vet J Suppl 1985; 3:23–24.
  4. Holtan DW, Houghten E, Silver M, Fowden AL, Ousey J, Rossdale PD. Plasma progestagens in the mare, fetus and newborn foal. J Reprod Fertil Suppl 1991; 44:517–528.[Medline]
  5. Squires EL, Ginther OJ. Follicular and luteal development in pregnant mares. J Reprod Fertil Suppl 1975; 23:429–433.
  6. Squires EL, Stevens WB, Pickett BW, Nett TM. Role of pregnant mare serum gonadotropin in luteal function of pregnant mares. Am J Vet Res 1978; 40:889–891.
  7. Martin JL, Saltiel A, Evans JW. Progesterone synthesis by different types of corpora lutea during the first 198 days of pregnancy. J Equine Vet Sci 1989; 9:84–87.
  8. Ginther OJ. Reproductive Biology of the Mare: Basic and Applied Aspects. Cross Plains, WI: Equiservices; 1992.
  9. Stewart F, Allen WR. The binding of FSH, LH and PMSG to equine gonadal tissues. J Reprod Fertil Suppl 1979; 27:431–440.
  10. Roser JF, Evans JW. Luteal luteinizing hormone receptors during the preovulatory period in the mare. Biol Reprod 1983; 29:499–510.[Abstract]
  11. Pineda MH, Ginther OJ, McShan WH. Regression of corpus luteum in mares treated with an antiserum against an equine pituitary fraction. Am J Vet Res 1972; 33:1767–1773.[Medline]
  12. Pineda MH, Garcia MC, Ginther OJ. Effect of antiserum against an equine pituitary fraction on corpus luteum and follicles in mares during diestrus. Am J Vet Res 1973; 34:181–183.[Medline]
  13. Kelly CM, Hoyer PB, Wise ME. In-vitro and in-vivo responsiveness of the corpus luteum of the mare to gonadotrophin stimulation. J Reprod Fertil 1988; 84:593–600.[Abstract]
  14. Watson ED, Colston M, Broadley C. LH and progesterone concentrations during diestrus in the mare and effect of hCG. Theriogenology 1995; 43:1325–1337.[CrossRef]
  15. Allen WR, Moor RM. The origin of the equine endometrial cups. I: Production of PMSG by fetal trophoblast cells. J Reprod Fertil 1972; 29:313–316.[Medline]
  16. Hamilton DW, Allen WR, Moor RM. The origin of equine endometrial cups III. Light and electron microscopic study of fully developed equine endometrial cups. Anat Rec 1973; 177:503–518.[CrossRef][Medline]
  17. Allen WR. Hormonal control of early pregnancy in the mare. Vet Clin North Am Large Anim Pract 1980; 2:291–302.[Medline]
  18. Squires EL, Garcia MC, Ginther OJ. Effects of pregnancy and hysterectomy on the ovaries of pony mares. J Anim Sci 1974; 38:823–830.
  19. Ginther OJ, First NL. Maintenance of the corpus luteum in hysterectomized mares. Am J Vet Res 1971; 32:1687–1691.[Medline]
  20. Squires EL, Wentworth BC, Ginther OJ. Progesterone concentration in blood of mares during the estrous cycle, pregnancy and after hysterectomy. J Anim Sci 1974; 39:759–767.
  21. Bergfelt DR, Pierson RA, Ginther OJ. Resurgence of the primary corpus luteum during pregnancy in the mare. Anim Reprod Sci 1989; 21:261–270.[CrossRef]
  22. Daels PF, Shideler S, Lasley BL, Hughes JP, Stabenfeldt GH. Source of oestrogen in early pregnancy in the mare. J Reprod Fertil 1990; 90:55–61.[Abstract]
  23. Daels PF, DeMoraes MJ, Stabenfeldt GH, Hughes JP, Lasley BL. The corpus luteum: a major source of estrogen during early pregnancy in the mare. J Reprod Fertil Suppl 1991; 44:501–508.[Medline]
  24. Daels PF, Chang GC, Hansen B, Mohammed HO. Testosterone secretion during early pregnancy in mares. Theriogenology 1996; 45:1211–1219.
  25. Al-Timimi I, Gaillard J, Amri H, Silberzahn P. Androgen synthesis and aromatization by equine corpus luteum microsomes. J Biol Chem 1989; 264:7161–7168.[Abstract/Free Full Text]
  26. Amri H, Silberzahn P, Al-Timimi I, Gaillard JL. Aromatase activity in the mare ovary during estrous cycle. Measurement of endogenous steroids and of their in vitro inhibitory effect. Acta Endocrinol (Copenh) 1993; 129:536–542.[Medline]
  27. Amri H, Gaillard JL, Al-Timimi I, Silberzahn P. Equine ovarian aromatase: evidence for a species specificity. Biochem Cell Biol 1993; 71:296–302.[Medline]
  28. Almadhidi J, Seralini G, Fresnel J, Silberzahn P, Gaillard J. Immunohistochemical localization of cytochrome P450 aromatase in equine gonads. J Histochem Cytochem 1995; 43:571–577.[Abstract]
  29. Daels PF, Albrecht BA, Mohammed HO. In-vitro regulation of luteal function in mares. Reprod Domest Anim 1995; 30:211–217.
  30. Daels PF, Ammon DC, Stabenfeldt GH, Liu IKM, Hughes JP, Lasley BL. Urinary and plasma estrogen conjugates, estradiol and estrone concentrations in nonpregnant and early pregnant mares. Theriogenology 1991; 35:1001–1017.
  31. Munro C, Stabenfeldt GH. Development of microtitre plate enzyme immunoassay for the determination of progesterone. J Endocrinol 1984; 101:41–49.[Abstract]
  32. Fortune JE, Eppig JJ. Effects of gonadotropins on steroid secretion by infantile and juvenile mouse ovaries in vitro. Endocrinology 1979; 105:760–768.[Medline]
  33. Shideler SE, Czekala NM, Benirschke K, Lasley BL. Urinary estrogens during pregnancy of the ruffed lemur (Lemur variegatus). Biol Reprod 1983; 28:963–969.[Abstract]
  34. Matteri RL, Roser JF, Baldwin DM, Lipovetsky V, Papkoff H. Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species. Domest Anim Endocrinol 1987; 4:157–165.[CrossRef][Medline]
  35. Daels PF, Jorge-De Moraes M, Stabenfeldt GH, Hughes JP. The effect of altrenogest on the development of secondary corpora lutea. Proc 12th Int Congr Anim Reprod 1992; 4:1855–1857.
  36. Allen WR. The immunological measurement of pregnant mare serum gonadotropin. J Endocrinol 1969; 43:593–598.
  37. Albrecht BA, MacLeod JN, Daels PF. Differential transcription of steroidogenic enzymes in the equine primary corpus luteum during diestrus and early pregnancy. Biol Reprod 1997; 56:821–829.[Abstract]
  38. Albrecht BA, Daels PF, MacLeod JN. Luteal cytochrome P450 17{alpha}-hydroxylase-17,20-lyase (P45017{alpha}) mRNA levels increase after the onset of chorionic gonadotropin secretion in pregnant mares. Biol Reprod 1996; 56(suppl 1):110.
  39. Albrecht BA, MacLeod JN, Daels PF. Cytochrome P450 aromatase mRNA levels decrease in equine luteal tissue after the onset of chorionic gonadotropin secretion. Biol Reprod 1995; 52(suppl 1):111.
  40. Albrecht BA, MacLeod JN, Daels PF. Expression of luteal cytochrome P450 aromatase (P450arom) before and after the onset of chorionic gonadotropin secretion in mares. J Reprod Fertil 1997; 19:43.
  41. Jarry H, Einspanier A, Kanngiesser L, Dietrich M, Pitzel L, Holtz W, Wuttke W. Release and effects of oxytocin on estradiol and progesterone secretion in porcine corpora lutea as measured by in vivo microdialysis system. Endocrinology 1990; 126:2350–2358.[Abstract]
  42. Maas S, Jarry H, Teichmann A, Rath W, Kuhn W, Wuttke W. Paracrine actions of oxytocin, prostaglandin F2{alpha}, and estradiol within the human corpus luteum. J Clin Endocrinol Metab 1992; 74:306–312.[Abstract]
  43. McLean MP, Miller JB. Steroidogenic effect of 17ß-estradiol on rabbit luteal cells in vitro: estrogen-induced maintenance of progesterone production. Biol Reprod 1985; 33:459–469.[Abstract]
  44. McLean MP, Miller JB. Lipoprotein-stimulated and estradiol-maintained progesterone secretion by dissociated rabbit luteal cells in vitro. Biol Reprod 1986; 34:642–654.[Abstract]
  45. Gibori G. The corpus luteum of pregnancy. In: Adashi EY, Leung PCK (eds.), The Ovary. New York: Raven Press; 1993: 261–317.
  46. Wuttke W, Jarry H, Pitzel L, Knoke I, Spiess S. Luteotrophic and luteolytic actions of ovarian peptides. Hum Reprod 1993; 8(suppl 2):141–146.
  47. Broadley C, Menzies GS, Bramley TA, Watson ED. Isolation of cell populations from the mare corpus luteum: comparison of mechanical and collagenase dissociation. J Reprod Fertil 1994; 102:7–15.[Abstract]
  48. Watson ED, Sertich PL. Secretion of prostaglandins and progesterone by cells from corpora lutea of mares. J Reprod Fertil 1990; 88:223–229.[Abstract]
  49. Condon WA, Ganjam VK, Kenney RM. Catecholamines and equine luteal progestagens. J Reprod Fertil Suppl 1979; 27:199–203.


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