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Regular Article |
a Laboratories of Theriogenology and
b Pathophysiology, National Institute of Animal Health, Tsukuba, Ibaraki 305-0856,
c Japan Department of Internal Medicine, Nippon Veterinary and Animal Science University, Musashino, Tokyo 180-8602, Japan
ABSTRACT
The release profile of GnRH in cerebrospinal fluid (CSF) and its correlation with LH in peripheral blood of ovary-intact heifers during the estrous cycle were investigated. A silicon catheter was placed into the third ventricle of six heifers using ultrasonography. During the mid-luteal phase, the heifers were injected with prostaglandin F2
to induce luteolysis. Surges of CSF GnRH (66.7 h after prostaglandin F2 administration) and peripheral LH (66.3 h) occurred simultaneously and were coincident with the onset of estrus (67.0 h). Duration of elevated GnRH concentration considerably overlapped with the estrous phase in each of the heifers. Mean pulse frequencies of both GnRH and LH were significantly higher during the proestrous and early luteal phases than during the mid-luteal phase, while mean concentration and pulse amplitude of both GnRH and LH were not different between these three phases. Of all the GnRH pulses identified, more than 80% were accompanied by an LH pulse during the proestrous and early luteal phases. However, the proportion of GnRH pulses that were coincident with an LH pulse during the mid-luteal phase decreased to 60%. The results clearly demonstrate that a dynamic (pulse) and longer-term (surge) changes of GnRH release into CSF are physiologically expressed during the estrous cycle in heifers, and the pattern of pulsatile GnRH secretion in heifers depends upon their estrous cycle.
GnRH, hypothalamus, ovulatory cycle
INTRODUCTION
Analysis of GnRH secretion in mammals provides useful information for understanding the central regulation of reproduction. The preceding secretion of GnRH from the hypothalamus into the hypophyseal portal blood is essential for LH secretion from the pituitary gland. Thus, direct measurements of GnRH in hypophyseal portal blood are the most accurate assessment of hypothalamic activity and success in rats [1], sheep [2, 3], young calves [4], and goats [5]. However, the complex anatomical architecture of the cranium presents a significant barrier to the practical application of this method in adult cattle.
GnRH is detectable in third ventricular cerebrospinal fluid (CSF), and the patterns of GnRH release into CSF correspond directly (or sometimes with a time delay) to that of serum LH in the rhesus monkey [6], rabbit [7], and sheep [3, 8]. Thus, monitoring of CSF GnRH may be an alternative system for assessing GnRH secretion. Recently, Gazal et al. [9] reported the detection of GnRH in third ventricular CSF of ovariectomized or intact but anestrous adult cattle. However, changes in patterns of CSF GnRH secretion during the estrous cycle of cattle are not still clear.
Technical and economical limitations have made the use of adult cattle impractical for experimental studies because the stereotaxic method, which is used for the neurosurgical approach, needs specific equipment. However, cattle have a great advantage in that changes in ovarian structures, such as follicular growth, ovulation, and growth and regression of corpus luteum, can be monitored in real time by transrectal ultrasonography [10]. This will lead to significant information on the central regulation of reproduction, such as how changes in GnRH secretion affect not only LH secretion from the pituitary but also changes in ovarian structures.
The present study reports a new technique for cannulating by transdural ultrasonography and sampling CSF from the third ventricle of heifers. The objective of the study was to characterize GnRH secretion during the estrous period and to determine how it relates to the preovulatory LH surge and estrous behavior in the ovary-intact heifer. We also investigated pulsatile GnRH secretion during the proestrous, early luteal, and mid-luteal phases of the estrous cycle.
MATERIALS AND METHODS
Animals
Sexually mature and estrous-cyclic Holstein heifers (n = 6; 24–36 mo old, 460–580 kg body weight) were used in the experiment. All animal-related procedures employed in this study were approved by the Institutional Care and Use Committee for Laboratory Animals of the National Institute of Animal Health (Protocol 88) and the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching.
Heifers were housed in stanchion stalls under natural conditions of sunlight and temperature. They were fed concentrates and hay that were formulated to meet a 0.6-kg daily gain based on the Japanese Feeding Standard for Dairy Cattle [11] and had free access to water. Follicular and luteal structures in ovaries were examined at 1- to 2-day intervals by transrectal ultrasonography as previously described [12]. Ultrasonography was performed using a probe with a 7.5 MHz transducer (UST-5821-7.5; Aloka, Tokyo, Japan) and a linear-array ultrasound scanner (SSD-2000; Aloka). At each examination, the diameter and location of each structure 
4 mm in diameter was recorded. When a heifer exhibited standing estrus to be mounted by another heifer or cow, it was determined to be in estrus.
Surgical Catheterization of the Third Ventricle
Insertion of third ventricle catheters in heifers using ultrasonography was performed during the luteal phase of the cycle (5–12 days after ovulation). Briefly, animals were sedated with xylazine (40 mg/animal, i.v.) after atropine (5 mg/animal, i.m.) was administered to prevent excessive salivation. The surgical site was prepared for sterile surgery. Heifers were then placed in ventral recumbency and intubated. Anesthesia was induced and maintained by a closed circuit system of isoflurane (2.0%–2.5% Forane; Dainippon Pharmaceutical, Osaka, Japan), nitrous oxide (3–4 L/min), and oxygen (4 L/min).
The center area in positioning of the surgery was calculated to be 3/4 of the distance between the poll and the caudal margins of the eye orbits from the poll on the supposed mid-saggital line. This positioning of the surgery was referred to the stereotaxic positioning described previously [13]. After surgical preparation, skin and muscle were reflected to expose the frontal bone. A hole approximately 2 x 3 cm was drilled at the target site through the frontal bones and sinus cavity. A probe with a 5 MHz transducer for brain surgery (UST-9104-5; Aloka) attached to a linear-array ultrasound scanner was placed on the dura and the third ventricle was visualized (Fig. 1). The probe attachment (MP-2458; Aloka) was fitted with the probe to accurately puncture the guide cannula. A 16-gauge stainless steel guide cannula with an occluding stylet was lowered to the third ventricle with the help of the puncture guide line on the ultrasound monitor. After removing the stylet and ascertaining that CSF flowed vigorously from the guide cannula, a silicone catheter (Radifocus GT Catheter III, 2.7 Fr; Terumo, Tokyo, Japan) filled with lacto-Ringer solution (Terumo) was threaded into the guide cannula. The tip of this catheter could be verified ultrasonographically because it was fitted with a platinum coil. The guide cannula was removed entirely, leaving only the silicon catheter in the third ventricle. The tip of the catheter was located as close to the bottom of the third ventricle as possible. The dura was covered with a gelatin sponge (Spongel; Yamanouchi Pharmaceutical, Tokyo, Japan). A polyvinyl chloride ring (40 mm inside diameter [i.d.] x 48 mm outside diameter [o.d.] x 25 mm height) was placed in the circumscribed area surrounding the catheter, and the ring and catheter were fixed to frontal bones with acrylic dental cement (GC repairsin; GC, Tokyo, Japan). The silicone catheter was cut to a length of 45–55 cm, and a blunted 20-gauge needle and a plug with a rubber cap (Intermittent Infusion Plug; Argyle, Nippon Sherwood, Tokyo, Japan) were connected to the end of the catheter. The catheter was coiled within the polyvinyl chloride ring and was retained with a screw cap to protect the catheter. After surgery, heifers were injected daily with antibiotics (10 ml Duopen; Tanabe Seiyaku, Osaka, Japan) for 3 days.
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Experimental Protocol
A period of at least 3 wk was allowed between cranial surgery and the start of an experiment, and estrus and ovulation were observed in each heifer once or twice during this period. At 2300 h at 10 days postovulation, heifers were injected with prostaglandin F2 (PGF2) as 25 mg of dinoprost (Panacelan Hi; Daiichi Pharmaceutical, Tokyo, Japan) to induce luteolysis. Follicular and luteal structures in the ovaries that were 4 mm in diameter were monitored daily by transrectal ultrasonography. The structures were monitored from 2 days before PGF2 administration to 7 days postovulation of the second estrous cycle after PGF2 administration. During this period, a peripheral blood sample was also collected daily from the jugular vein. The samples were kept on ice and taken to the laboratory within 30 min. Plasma was separated and stored at -20°C until assayed for estradiol-17ß and progesterone.
To determine whether a GnRH surge in third ventricular CSF could be observed in estrous-cyclic heifers and whether it was associated with estrous behavior, CSF samples were collected at 1-h intervals simultaneously with jugular blood samples for 36 h between 54 h (0500 h) and 90 h (1700 h) after PGF2 administration. When a CSF sample was collected, the screw cap was opened and the rubber plug head of the catheter was sterilized with alcohol. To collect CSF samples for monitoring the surge, a 26-gauge needle connected to a 1-ml syringe was inserted into the plug and CSF was gently drawn up into the syringe. After removing the first 250–300 µl of CSF that had remained in the catheter, 1 ml of CSF was collected with the syringe. The samples were immediately placed into a glass tube containing 2 ml of methanol and were kept at 4 °C for less than 6 h. After centrifugation (1500 x g; 15 min at 4°C) to extract proteins, the supernatant was poured into a glass tube and dried in a vortex evaporator (Labconco, Lenexa, KS). Extracted samples were stored at -20°C until assayed for GnRH. Jugular blood was collected via an indwelling jugular polyethylene catheter (14-gauge; Nippon Zenyaku Kogyo, Koriyama, Japan) inserted at least 36 h before the start of sampling for the surge. After collection, the blood samples were kept for 6–18 h at room temperature. Serum was separated and stored at -20°C until assayed for LH. Standing estrus was also checked at 6-h intervals for 36 h between 54 and 90 h after PGF2 administration.
To determine whether GnRH pulses in the CSF were coincident with LH pulses, CSF samples were collected at 10-min intervals simultaneously with jugular blood samples for 6 h from 1100 to 1700 h during the proestrous, early luteal, and mid-luteal phases (2, 8, and 15 days after PGF2 administration, respectively). The times corresponded to 3 days before, and 3 and 10 days after ovulation, respectively. To collect CSF samples for determining pulsatile secretion of GnRH, a rubber-capped plug was removed from the end of the catheter. The catheter was connected to approximately 120 cm of Teflon tubing (0.25 mm i.d. x 1.5 mm o.d.; GL Sciences, Tokyo, Japan) with a male Luer-lock fit. The Teflon tubing was connected to approximately 25 cm of peristaltic tubing (1 mm i.d. x 3 mm o.d.; Iuchi Seieido, Tokyo, Japan), which was threaded through a peristaltic pump (SJ-1211L; Atto, Tokyo, Japan). The other end of the peristaltic tubing was connected to approximately 40 cm of the same Teflon tubing. The total volume of the tubing was approximately 700–800 µl. The end of the Teflon tubing was set above a glass tube containing 2 ml of methanol on the automatic fraction collector (Frac-100; Pharmacia Biotech, Uppsala, Sweden). CSF fractions were collected every 10 min at a collection rate of 100 µl/min. After all the CSF samples were collected, they were centrifuged (1500 x g; 15 min at 4°C) to remove proteins. The supernatant was poured into a glass tube and dried in a vortex evaporator. Dried glass tubes were stored at -20°C until assayed for GnRH. Jugular blood was collected via an indwelling jugular polyethylene catheter inserted at least 16 h before the start of sampling for the pulse. A blood sample was collected at the same time at the end of each CSF fraction period. The blood samples were kept overnight at room temperature. Serum was separated and stored at -20°C until assayed for LH.
Hormone Assays
CSF samples were assayed for GnRH after extraction using the method of Tanaka et al. [5] with a slight modification. Buffer (250 µl) was added to the dried extract, from which two 100-µl aliquots were used to determine the GnRH concentration. As a primary antibody, CRR11B71 GnRH antiserum (provided by Dr. Ramirez, University of Illinois) was used at a final dilution of 1:150 000. Assay sensitivity for GnRH averaged 0.21 pg/tube. The intraassay and interassay coefficients of variation (CV) were 9.8% and 7.6%, respectively. Blood samples were assayed for LH in duplicate 100-µl aliquots of serum by the radioimmunoassay (RIA) method described previously [12]. Assay sensitivity for LH averaged 0.03 ng/tube. The intraassay and interassay CVs were 9.1% and 10.9%, respectively. Plasma concentrations of estradiol-17ß and progesterone were determined using a previously described RIA [12]. Assay sensitivities for estradiol-17ß and progesterone averaged 0.21 and 1.04 pg/tube, respectively. The intraassay and interassay CV were 9.6% and 11.0%, respectively for estradiol-17ß, and 15.5% and 12.2% for progesterone.
Data Analysis
The onset of the GnRH and LH surges were defined by the method described previously [8]. Pulses of GnRH and LH were analyzed with the computer algorithm, PULSAR [14, 15]. The cutoff criteria consisted of the following: 3.0, 1.0, and 0.4 times SD of the assay for one-, two-, and three-point peaks. For each pulse, mean overall and baseline hormone levels, mean pulse frequency, and mean pulse amplitude were determined by the program.
Synchrony between the GnRH and LH pulses was assumed when the maximal GnRH and LH concentrations obtained during a pulse occurred within one sample (10 min) of each other. More stringent requirement that a GnRH pulse either precede or coincide exactly with the LH pulse was not warranted because each GnRH value represents the concentration of a CSF sample collected continuously during a 10-min period passing through the catheter with approximately 700–800 µl of internal volume, whereas each LH value is the concentration of a blood sample collected intermittently at the end of a 10-min period.
For all experiments, the results are expressed as mean ± SEM unless otherwise stated. Data were statistically analyzed with the StatView 5 (SAS Institute Inc., Cary, NC) software package. The onset of the GnRH and LH surges were compared using Student's paired t-test. For pulsatile GnRH or LH during each of the proestrous, early, and mid-luteal phases, differences in mean hormone level, pulse frequency, pulse amplitude, and percentage of synchrony between GnRH and LH were analyzed by one-way ANOVA. When the ANOVA showed a significant effect for the phase, phases were compared by Fisher's PLSD test. The amplitude of GnRH pulses with and without coincident LH pulses were compared by Student's paired t-test. A P value less than 0.05 denoted the presence of a statistically significant difference.
RESULTS
General
We were able to collect third ventricular CSF from all six heifers used in this study throughout the experimental period and no aberration in the estrous cycle was observed after cranial surgery. Three of six animals maintained functional catheters for more than 1 yr, except three heifers that were slaughtered at 2–3 mo after the end of the experiment. The changes in the ovarian structures, plasma concentrations of estradiol-17ß, and progesterone during the experimental period are shown in Figure 2. Standing estrus was started at 3 days after PGF2 administration. The ovulation was observed at 5 days after PGF2 administration. After ovulation, the corpus luteum that developed was maintained for 7–10 days and then regressed. The mean length of the estrous cycle for all heifers following PGF2 administration was 21.5 ± 1.4 days. During the estrous cycle, two follicular waves were observed in two of the heifers and three follicular waves were observed in the other four heifers. No endocrine aberration for estradiol-17ß and progesterone was observed.
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Estrous Behavior and Surges of GnRH and LH
The patterns of GnRH and LH for each of the six heifers treated with PGF2 are shown in Figure 3. Standing estrus and preovulatory surges of GnRH in CSF and LH in serum were detectable in all six heifers. The onset of standing estrus was observed at 67.0 ± 2.6 h after PGF2 administration and its duration was 14.0 ± 1.4 h. Concentrations of GnRH in CSF and LH in serum increased from their baseline level (6.0 ± 0.3 pg/ml and 2.2 ± 0.3 ng/ml, respectively) concomitant with the onset of standing estrus. Peak values of GnRH and LH averaged 19.8 ± 6.8 pg/ml and 41.2 ± 8.4 ng/ml, respectively. The onset of LH surge (66.3 ± 2.1 h after PGF2 administration) coincided with the onset of GnRH surge (66.7 ± 1.9 h after PGF2 administration). Duration of sampling was adequate for characterizing the entire LH surge in all cases (10.5 ± 0.8 h), but GnRH surges extending beyond those for LH were not fully characterized in some heifers by our sampling regimen. However, duration of elevated GnRH concentration considerably overlapped the estrous phase in each of heifers.
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Correlation of CSF GnRH Pulses and Peripheral LH Pulses in Estrous Cycle
All of the six heifers showed a pulsatile pattern of secretion of GnRH into CSF of the third ventricle during the proestrous, early luteal, and mid-luteal phases. Mean concentrations of estradiol-17ß and progesterone in plasma on the day that intensive sampling to detect pulses was made were 7.2 ± 1.0 pg/ml and 0.5 ± 0.1 ng/ml (proestrous), 4.3 ± 1.0 pg/ml and 1.8 ± 0.1 ng/ml (early luteal), and 2.3 ± 0.4 pg/ml and 6.5 ± 0.4 ng/ml (mid-luteal), respectively (Fig. 2). Representative patterns of GnRH and LH secretion in each of three heifers during the proestrous, early luteal, and mid-luteal phases are shown in Figure 4. Mean pulse frequencies of both GnRH and LH were significantly greater (P < 0.05) during the proestrous and early luteal phases than during the mid-luteal phase (Table 1). However, mean pulse amplitude and mean overall and baseline concentrations of both GnRH and LH were not different between these three phases. Of all the GnRH pulses identified, >80% were coincident with a pulse of LH during the proestrous and early luteal phases. In contrast to these two phases, the proportion of GnRH pulses coincident with an LH pulse during the mid-luteal phase decreased by 23%–25% and was significantly lower than that during the early luteal phase (P < 0.05). Approximately 80% of all the LH pulses identified were coincident with a GnRH pulse during all phases. Finally, of all the GnRH pulses detected, the amplitude of GnRH pulses without coincident LH pulses (n = 25; 1.3 ± 0.1 pg/ml) was significantly less (P < 0.01) than that of GnRH pulses with coincident LH pulses (n = 82; 2.2 ± 0.2 pg/ml).
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DISCUSSION
Our results clearly demonstrated that a preovulatory increase of GnRH secretion into third ventricular CSF occurs in estrous-cyclic heifers. This GnRH surge began not only coincident with the preovulatory LH surge, but also with the onset of standing estrus. Moreover, pulsatile secretion of GnRH in heifers during the proestrous, early luteal, and mid-luteal phases can be detected by frequent sampling of CSF, and LH pulses during these phases are synchronized with GnRH pulses.
In the present study, we have applied a new technique in which ultrasonography is used for catheterization of bovine third ventricle. In this study we left only the silicone catheter in the cranium without a stainless steel guide cannula. The silicone catheter is made of a very pliable material that is highly biocompatible. We believe that the use of this catheter resulted in less tissue damage and a lower risk of infection than occurs with methods that leave a guide cannula in the cranium. In the present study, catheters remained functional for more than 1 yr in some animals. The length of the estrous cycle and the main characteristics of changes in ovarian structures (ovulation, development, and regression of corpus luteum and dominant follicle, and number of follicular waves) after PGF2 treatment in this study were similar to those reported previously in cattle on natural or PGF2-induced estrous cycles [10, 16–21]. Moreover, changes of estradiol-17ß and progesterone concentrations in peripheral blood during the estrous cycle seemed to be normal [20, 21]. Thus, this catheterization method should be useful for sampling the third ventricular CSF in adult cattle.
In a recent study, Gazal et al. [9] reported the detection of estradiol-induced GnRH surges in CSF of ovariectomized cattle, but to our knowledge this is the first report of this phenomenon in ovary-intact and estrous-cyclic heifers. The mean duration of LH surge in this study was similar to that of natural or PGF2-induced LH surges [16, 22–24], although it has been reported that the mean duration of an estradiol-induced LH surge in ovariectomized cattle in which CSF was sampled was shorter than that of the natural LH surge [9]. The general pattern of GnRH release during the LH surge was similar to that of estradiol-induced GnRH surges in CSF of ovariectomized cattle [9] and ewes [3, 8]. Mean peak concentration of the GnRH surge in CSF was also similar to that described in ovariectomized cattle [9], but it was lower than that reported in ewes [3, 8]. The mean preovulatory peak concentration of LH was similar to the peak concentrations reported previously in cattle [16, 20, 22–25]. Because pulsatile secretion of GnRH in CSF was detectable in the present study, these findings indicate that the amplitude of GnRH surge in CSF may be lower in cattle than in ewes.
In our study, standing estrus was also examined. The onset of standing estrus after PGF2 treatment in this study was similar to that reported previously [24–27]. The onset of GnRH surge in CSF coincided with not only the onset of LH surge, but also the onset of standing estrus. Furthermore, duration of rise of GnRH concentration seemed to overlap with the presumed estrous phase in each of heifers. It is known that GnRH acts as a neurotransmitter, a neuromodulator, or both in the central nervous system and that it acts on the pituitary gonadotropes as well [28]. In female rats, GnRH can facilitate lordosis behavior [29, 30] and anti-GnRH injected into the mid-brain central gray [31] or the lateral ventricles [32] caused a blockage or reduction of the behavior. Moreover, GnRH influences sexual behavior in monkeys [33] and horses [34]. The present results suggest that GnRH also influences a bovine sexual behavior, standing estrus. In addition, one of the functions of GnRH in CSF may be to modulate reproductive behavior because it has been demonstrated that GnRH in CSF played no role in modulating gonadotropin secretion in the ovariectomized ewe [35]. Whether GnRH and specially CSF GnRH modulates sexual behavior in cattle should be investigated to clarify the function of CSF GnRH.
The CSF withdrawal rate used in this study (100 µl/min) for the analysis of pulsatile GnRH secretion did not appear to attenuate pituitary function. This is consistent with observations in ewe [8] in which the same sampling rate was used but in which the CSF volume is much smaller than in cattle. The characteristics of LH pulses in this study were similar to those during natural or PGF2-induced estrous cycles reported previously [36–38]. Ovarian steroids act upon the central nervous system to influence the hypothalamic release of GnRH and upon the anterior pituitary response to GnRH by their feedback effects [36, 39–41]. In cows, modulation of LH secretion by ovarian steroids is complicated because these steroids act synergistically to maintain the pattern of LH secretion within the range observed during the estrous cycle [39, 42]. Significantly fewer GnRH and LH pulses were measured during the mid-luteal phase than during the proestrous or early luteal phase in the present study. These results suggest that the steroid level during the mid-luteal phase (low circulating concentration of estradiol and high circulating concentration of progesterone) reduced the hypothalamic release of GnRH in heifers.
Percentages of the GnRH pulses in CSF that were accompanied by an LH pulse during the proestrous and early luteal phases (82%–85%), are similar to the percentages previously observed (79%–87%) in rhesus monkeys, ewes, and cows [3, 6, 8, 9]. The GnRH pulses that were not accompanied by an LH pulse have been documented in sheep, rat, rhesus monkey, and cattle utilizing various procedures for assessment of hypothalamic GnRH secretion [2, 4, 6, 43–45]. It has been reported that small-amplitude GnRH pulses in hypophyseal portal blood did not induce a coincident LH pulse in ewes [46, 47]. While portal GnRH pulses tended to have greater amplitude compared with CSF GnRH pulses in ewes [3], even the amplitude of CSF GnRH pulses without coincident LH pulses was significantly smaller than the amplitude of GnRH pulses with coincident LH pulses in heifers of the present study.
In the present study, the number of GnRH pulses that occurred in the absence of a detectable LH pulse was dramatically increased during the mid-luteal phase, and the proportion of GnRH pulses coincident with an LH pulse during the mid-luteal phase was reduced to 60%. The incidence of GnRH pulses that occurred in the absence of a detectable LH pulse may fluctuate during the estrous cycle in heifers. Ovarian steroid levels during the mid-luteal phase may not only reduce the hypothalamic release of GnRH accessed by pulse frequency, but they may also directly suppress responsiveness of pituitary gonadotropes to GnRH in ovary-intact heifers. In fact, progesterone reduces the number of receptors for GnRH in the pituitary by down-regulating mRNA encoding the receptor for GnRH [48, 49].
In summary, these data demonstrate that changes in GnRH concentrations in CSF of the heifer during the estrous cycle could be used as an index of the GnRH neuroendocrine system. GnRH neurosecretion appears to be regulated by feedback effects of ovarian steroids during the estrous cycle. In adult female cattle in which ovarian structures are traceable in real time, analysis of GnRH in CSF may provide a more complete understanding of endocrine network on hypothalamus-pituitary-gonad axis. Furthermore, this technique for sampling CSF from bovine third ventricle, which is large compared with the third ventricles of rabbits, rhesus monkeys, and sheep may be useful for understanding changes in neurohumoral factors in CSF because multiple factors can be analyzed simultaneously.
ACKNOWLEDGMENTS
We thank Dr. T. Tanaka (Tokyo University of Agriculture and Technology) for his useful advice regarding GnRH assay, Dr. V.D. Ramirez (University of Illinois) for providing antiserum to GnRH, Dr. L.E. Reichert (Albany Medical College of Union University) for providing bovine LH, and Dr. G.D. Niswender (Colorado State University) for providing antisera to estradiol-17ß and progesterone.
FOOTNOTES
First decision: 22 August 2000.
1 Supported by a Brain Atlas Program grant from the Ministry of Agriculture, Forestry and Fisheries, Japan. 
2 Correspondence: Koji Yoshioka, Laboratory of Theriogenology, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. FAX: 81 298 38 7880; kojiyos{at}niah.affrc.go.jp
Accepted: September 20, 2000.
Received: July 31, 2000.
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