# Gonadotropins in lactating sows exposed to long or short days during pregnancy and lactation: serum concentrations and ovarian receptors

A.Y. Kermabon, Armelle Prunier, Jean Djiane, Roland Salesse

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A.Y. Kermabon, Armelle Prunier, Jean Djiane, Roland Salesse. Gonadotropins in lactating sows exposed to long or short days during pregnancy and lactation: serum concentrations and ovarian receptors. Biology of Reproduction, 1995, 53, pp.1095-1102. hal-02706943

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# Gonadotropins in Lactating Sows Exposed to Long or Short Days during Pregnancy and Lactation: Serum Concentrations and Ovarian Receptors<fnref n="1" />

Anne-Yvonne Kermabon,<fnref n="2" />,³ Armelle Prunier,⁴ Jean Djiane,³ and Roland Salesse<fnref n="2" />,³

*Laboratoire de Biologie Cellulaire et Moléculaire,³ Institut National de la Recherche Agronomique*
78352 Jouy-en-Josas Cedex, France

*Station de Recherches Porcines,⁴ Institut National de la Recherche Agronomique, 35590 St. Gilles, France*

## ABSTRACT

The objective of this study was to determine the influence of day length on gonadotropin profiles and the expression of their ovarian receptors in lactating sows. Primiparous Large White gilts were exposed to either a gradual increase (from 12 to 16 h/day, LONG treatment, n = 13) or decrease (from 12 to 8 h/day, SHORT treatment, n = 12) in photoperiod during gestation. Weaning occurred at Day 21 of lactation. All 4 sows that were submitted to the SHORT light duration and checked for postpartum estrus demonstrated an estrus by 10 days postwearing in contrast to 2 of 5 sows submitted to the LONG light duration (*p* < 0.05). In the remaining 16 sows, day length had no significant effect on the number of LH pulses or on mean or basal concentrations of gonadotropins (FSH and LH) or estradiol-17β measured at Day 20 of lactation. Ovarian receptors for gonadotropins and prolactin (PRL) and their mRNA were measured through use of receptor-binding and slot-blot analyses, respectively, at Day 21 of lactation in these 16 sows. A LONG photoperiod duration had no influence on receptor number, binding, or affinity, but it significantly increased LH receptor mRNA levels (*p* < 0.05). However, FSH receptor mRNA levels were similar in the two groups of sows. Plasma LH concentration was positively related to LH and FSH receptor content but not to their cognate mRNA levels. Plasma concentration of FSH was positively related to the level of its own receptor mRNA as well as to that of the PRL receptor mRNA. Although the LONG day length may have delayed the return to estrus, there was no effect on gonadotropin secretion. Our results show an effect of photoperiod only on the level of LH receptor mRNA. We suggest that not all transcripts of the LH receptor are translated and that nontranslatable mRNAs accumulate in ovaries of sows exhibiting delayed estrus.

## INTRODUCTION

European wild boars are seasonal breeders, and the fe-
males exhibit anestrus during summer [1]. This phenome-
non has waned in domestic pigs, even though a tendency
for summer infertility exists that has considerable economic
significance [2]. Sows usually exhibit estrus 4–8 days after
weaning, but this interval is often prolonged in summer,
particularly in primiparous sows [3–6]. High ambient tem-
peratures and long light duration in summer may explain
this delay [2, 7, 8]. Actually, high ambient temperatures dur-
ing lactation prolong the weaning-to-estrus interval, and
this effect could be mediated, at least in part, through a
decrease in food intake [7, 9]. Increasing the light duration
progressively during gestation in order to simulate the nat-
ural day length transition also increases the weaning-to-es-
trus interval [8, 10].

The endocrine mechanisms underlying the influence of
photoperiod on the return to estrus in sows after weaning are
poorly understood and are probably of multifactorial origin.
The 4–8-day time lapse between weaning and estrus is similar
to the length of the follicular phase of the estrous cycle in gilts;
this suggests that follicular recruitment for preovulatory
growth normally occurs immediately after weaning. A drop

in plasma prolactin (PRL) concentrations and an increase in
LH pulsatility and FSH levels are generally observed at wean-
ing and probably allow and/or stimulate follicular recruitment
[11, 12]. Thus, delayed estrus after weaning in summer may
result from an inadequate responsiveness of the follicles at
the time of weaning, which itself may result from insufficient
gonadotropin or excessive PRL secretions during lactation.
Some investigators have compared LH secretion in female
pigs at different seasons, but they were unable to distinguish
between the influences of photoperiod, temperature, and nu-
trition [6, 13, 14] on females submitted abruptly to 8 or 16 h
of light per day [15]. LH pulsatility did not change with season
or light duration in intact lactating sows, intact sows after
weaning, or ovariectomized females. No influence of light du-
ration on mean plasma LH concentration was observed in in-
tact lactating or ovariectomized females [15]. However, LH
concentration is lower in sows shortly after weaning in sum-
mer as compared to winter [6], and the influence of season
in ovariectomized females is controversial (higher LH con-
centration in summer [13]; no effect [14]). The effect of pho-
toperiod on FSH secretion has been less well studied. Mean
plasma FSH levels do not seem to be influenced by photo-
period in lactating sows [8] and are higher in summer than in
winter in sows shortly after weaning, probably in relation to
lower ovarian negative feedback tone [6]. Plasma concentra-
tions of PRL are higher in summer than in winter in ovariec-
tomized gilts, but the difference is less pronounced for cyclic
gilts [16]. Abrupt variation in light duration has no influence
on PRL levels in intact lactating or ovariectomized females [17].

<footnote>Accepted June 12, 1995.</footnote>

<footnote>Received December 5, 1994.</footnote>

<footnote n="1">¹This study was supported by grants from the Institut National de la Recherche Agronomique, France.</footnote>

<footnote n="2">²Correspondence: A.Y. Kermabon/Roland Salesse, Laboratoire de Biologie Cellulaire et Moleculaire, Institut National de la Recherche Agronomique, Batiment des Biotechnologies, 78352 Jouy-en-Josas Cedex, France. FAX: 33 (1)34.65.22.73.</footnote>
<figure><img src="image_2.png" /><figcaption>FIG. 1. Experimental design. Sows were bred at 10–13 mo of age and exposed to one of two light regimens that mimic variations occurring during spring (group LONG, n = 13) and autumn (group SHORT, n = 12). Weaning occurred at Day 21 of lactation.</figcaption></figure>

To our knowledge, the influence of season and photoperiod on follicular growth and responsiveness to gonadotropin stimulation has not been described in cyclic and postpartum female pigs.

The objective of the present study was to determine the influence of day length on the return to estrus after weaning and on the hormonal profiles of FSH and LH as well as on the expression of the ovarian receptors for FSH, LH, and PRL at the mRNA and protein levels in lactating sows. The fluctuation of these receptors and of their cognate mRNAs could provide insight into the ability of the ovary to respond to pituitary gonadotropins after weaning.

## MATERIALS AND METHODS

### *Animals and Experimental Design*

From 240 days of age, 25 pure Large White gilts were reared under controlled light conditions that have been previously described [8]. These females were bred at 10–13 mo of age and exposed to one of two light regimens that mimic variations occurring during spring (group LONG: 16 h of light, n = 13) and autumn (group SHORT: 8 h of light, n = 12) (Fig. 1). Ten days before expected farrowing, females were transferred to conventional farrowing crates. Three sows were eliminated from the experiment because of lack of pregnancy (n = 2) or intestinal hemorrhage (n = 1). Cross-fostering was performed within 48 h after birth in order to limit variability in litter size. Sows suckled 8–11 piglets throughout the period of study. Conventional feeding of the sows and management of the piglets were carried out as described previously [8].

Indwelling jugular cannulae were inserted surgically in 8

sows per group at 11 or 12 days after farrowing. To determine LH and FSH profiles, blood samples were drawn every 10 min between 0900 h and 1500 h on Day 20 of lactation.

The 16 sows used for the endocrine study were slaughtered (slaughterhouse of Station of Recherches Porcines, INRA St. Gilles, France) the day after blood sampling (Day 21 of lactation). Slaughter occurred less than 30 min after weaning. Piglets of the remaining sows were weaned on the same day, and sows were checked daily for estrus with a mature boar (group SHORT: n = 4; group LONG: n = 5). One blood sample was collected by venipuncture from sows not observed to be in estrus within 12 days after weaning, for determination of progesterone in order to ensure that ovulation had not occurred. Ovaries of slaughtered sows were removed and weighed, and the diameter of the largest follicle in each ovary was measured. Ovaries were then placed for 20 sec in cold isopentane (−70°C), snap-frozen in liquid nitrogen, and stored at −80°C until further analysis.

To obtain positive and negative controls in the mRNA analyses, portions of mammary gland and lung were collected and frozen in the same way. Testes were obtained by castration of Large White boars at 21 days of age, since at this stage the testes have substantial levels of gonadotropin receptors [18]. After removal of the epididymides, testes were prepared in the same way as the ovaries.

### *Hormonal Assays*

Concentration of LH was measured in all samples collected during the sequential sampling period. The concentration of FSH in hourly samples was quantified, and estradiol-17β was measured in the last sample collected. Samples were analyzed in duplicate within a single assay. Double-antibody RIAs were used for LH and FSH as previously described [19]. Highly purified porcine gonadotropins were used for standard curves and [¹²⁵I]-labeling. Porcine (p) LH used for the standard preparations was provided by UCB-Bioproducts (Brussels, Belgium). The biological activity of LH was 0.85X NIH LH S19. Unlabeled pFSH, a gift from Dr. Combarnous (INRA Nouzilly, France), had a biological activity of 81X NIH-FSH-P1. Specific anti-porcine LH serum was a gift from Dr. Camous (INRA, Jouy en Josas, France), and specific anti-porcine FSH was provided by UCB-Bioproducts. The minimum detection limit was 0.5 ng/ml for LH and FSH; intraassay coefficients of variation were 8.6% at 0.64 ng/ml for LH and 8.4% at 2.3 ng/ml for FSH. In the sample taken after weaning, a direct RIA was performed [20] to determine whether the progesterone concentration was higher than 5 ng/ml, which would indicate that ovulation had occurred. For estradiol, 2 ml of plasma was extracted with cold diethyl ether, and the assay was performed as previously described [21]. The minimum detection limit was 5 pg/ml estradiol, and the intraassay coefficient of variation was 5% at 410 pg/ml.
## Receptor Determinations

Ovarian membranes (microsomes) were prepared individually as previously described [22]. Briefly, one ovary from each sow was homogenized using an Ultraturrax (Polytron, pT10-35 340, Kinematica Ag, Switzerland) homogenizer at 4°C in 3 ml/g homogenization buffer (10 mM Tris-HCl [pH 7.4], 3.8 mM NaCl, 3 mM KCl, 1 mM PMSF, 1 mM benzamidine, and 0.04% NaN₃). The homogenate was centrifuged for 15 min at 500 × g at 4°C, and the supernatant was centrifuged for 90 min at 100 000 × g at the same temperature. The resulting pellet was resuspended in the homogenization buffer, and a fraction of the membrane preparation was assayed for protein content by the method of Lowry et al. [23]. The microsomes were stored at -20°C until use.

Purified hCG (a generous gift from Dr. R. Canfield, NIH, Bethesda, MD) was labeled with ¹²⁵I using iodogen as catalyst [24]. Ovine (o) PRL (NIDDK oPRL-16; 30.5 IU/mg) was kindly supplied by the National Hormone and Pituitary Program (NIH, Bethesda, MD) and was iodinated using chloramine T by the method of Greenwood et al. [25] as modified by Kelly et al. [26]. The labeled hormones were purified on a PD-10 Sephadex G-25 column (Pharmacia, St. Quentin en Yvelines, France), and the specific activity of the resulting tracer was 2200 Ci/mmol (oPRL) and 1500 Ci/mmol (hCG). The same protocol was followed for the determination of FSH receptors, except that the tracer [¹²⁵I]-human (h) FSH (2500-5600 Ci/mmol, used at 100 000 cpm/tube) was purchased from NEN (Les Ulis, France). Highly purified pFSH was used for nonspecific binding determination.

Specific binding of [¹²⁵I]-oPRL, [¹²⁵I]-hCG, and [¹²⁵I]-hFSH to membranes (1 mg protein) prepared from each ovary was determined in triplicate. Total binding was determined in the presence of 40-100 nM [¹²⁵I]-oPRL, -hCG, or -hFSH; nonspecific binding was measured in the presence of a large excess (1000-fold) of unlabeled oPRL, hCG (Chorulon, Intervet, Angers, France), or highly purified pFSH. After 16 h of incubation at 20°C, the incubations were stopped with 3 ml of ice-cold 10 mM Tris-HCl (pH 7.4) buffer for both hCG and hFSH and with 3 ml of 25 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 0.1% BSA for oPRL. Bound and free hormones were separated by centrifugation at 3500 × g at 4°C for 20 min. The supernatant was aspirated and the radioactivity of the membrane pellet was determined. Specific binding was calculated (nonspecific binding subtracted from total binding) and divided by total radioactivity to yield percentage of binding.

Scatchard analysis on randomly selected animals was performed as previously described [24, 27] by incubating in duplicate 100 µl of ovarian homogenate (1 mg of protein) with 150 000 cpm [¹²⁵I]-hCG (about 25 fmol) together with 100 µl of serial dilutions (0.1-1000 ng/tube) of unlabeled hCG (NIH, batch CR121) overnight at room temperature in

a total volume of 0.4 ml. The reactions were stopped as described above. The same protocol was followed for determination of FSH receptors, except that the tracer [¹²⁵I]-hFSH was used at 75 000 cpm/tube and highly purified and crude pFSH (gifts of Dr. Y. Combarnous) was used for isotopical dilution (0.1-500 ng/tube) and nonspecific binding determination, respectively.

## RNA Preparations

Total RNA was isolated from the second ovary of each animal according to the procedure of Chomczynski and Sacchi [28] as modified by Puissant and Houdebine [29]. Approximately half of the ovary from each sow was homogenized individually in 3 vol (w/v) of 4.2 M guanidinium thiocyanate (GuSCN; Fluka, Biochemicka, Buchs, Switzerland) solution containing 25 mM sodium citrate, 0.5% n-lauroyl-sarcosine (w/v), and 0.1 M β-mercaptoethanol. To this mixture, 0.1 vol of 2 M sodium acetate (pH 4) was added, and a first extraction with 1 vol phenol (water-saturated) and 0.2 vol chloroform (chloroform:iso-amyl alcohol, 49:1) was performed. The mixture was placed on ice for 15 min and centrifuged for 30 min at 3000 × g; the RNA in the aqueous phase was then precipitated by addition of 1 vol of isopropanol overnight at -20°C. The RNA pellets were recovered by centrifugation (30 min, 3000 × g, 4°C), rinsed with 70% ethanol, and dissolved in sterile water. RNA was precipitated by the addition of 6 vol of 4 M LiCl and kept at 4°C. After 4 h, the RNA pellets were recovered by centrifugation (12 000 × g, 30 min, 4°C) and rinsed with 70% ethanol; the dried pellets were then dissolved in 1 ml of a 10 mM Tris-HCl buffer (pH 7.5), 1 mM EDTA, 0.5% SDS solution (w/v) and ethanol-precipitated at -20°C in the presence of 0.2 M sodium acetate (pH 4). The RNA pellets were recovered by centrifugation and dissolved in 500 µl of a solution containing 10 mM Tris-HCl (pH 7.5), 0.12 M NaCl, 1 mM EDTA, and 0.5% SDS (w/v). The RNA was quantified by optical density measurements at 260 nm and distributed into aliquots of 20 and 40 µg. The RNA samples were kept in a precipitated form at -20°C.

## Complementary DNA Probes

All cDNA fragments were purified free of vector DNA by suitable endonuclease (purchased from Boehringer-Mannheim, Meylan, France) digestion and agarose gel electrophoresis. A 0.6-kb fragment (nt 344 to 944) coding for a portion of the rabbit PRL receptor cDNA [30] and a 2.2-kb cDNA fragment coding for the entire pLH receptor [31] and a mouse β-actin cDNA [32] were used. The FSH receptor template was a 0.8-kb fragment of the cDNA coding for the hormone-binding domain of the pFSH receptor cloned in our laboratory (Remy et al., unpublished experiments). Each probe was labeled by random priming by means of
<table><caption>TABLE 1. Wet weight of both ovaries and diameter of the largest superficial follicle per sow.</caption><thead><tr><th></th><th>Group LONG</th><th>Group SHORT</th></tr></thead><tbody><tr><td>Ovarian weight (g)</td><td>9.1 ± 0.9</td><td>10.5 ± 0.6</td></tr><tr><td>Follicle diameter (mm)</td><td>4.0 ± 0.3</td><td>4.4 ± 0.2</td></tr></tbody></table>

Values are mean ± SEM; n = 8.

the Amersham Multiprime kit (Amersham, Les Ulis, France) with [α³²P]dCTP (3000 Ci/mmol) for 3 h at room temperature, to specific activities of approximately 1 × 10⁹ cpm/µg. Unincorporated nucleotide triphosphates were eliminated through use of a QUIAGEN-tip 5 column (DIAGEN, Düsseldorf, Germany).

### RNA Slot-Blot Analyses

For slot-blot analyses, RNA from each animal was individually analyzed at three different concentrations. Briefly, 40 µg of ethanol-precipitated RNA was centrifuged (30 min, 12 000 × g, 4°C), washed with 70% ethanol, dried, and dissolved in 100 µl double-strength SSC (single-strength SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) with 7% formaldehyde (v/v). The RNA was denatured and then brought to a vol of 1 ml with 20-strength SSC; 2, 4, and 8 µg of each RNA preparation were deposited to Hybond C-extra nitrocellulose by means of a MINIFOLD II apparatus (Schleicher and Schuell, CERA-LABO, Ecquevilly, France) to triplicate filters. Filters were baked for 2 h at 80°C and then prehybridized in a solution consisting of 50% (v/v) deionized formamide, 5-strength Denhardt's solution (single-strength Denhardt's = 0.02% [w/v] each of BSA, Ficoll, and polyvinylpyrrolidone), 5-strength SSPE (single-strength SSPE = 1.15 M NaCl, 0.01 M NaH₂PO₄, 1 mM EDTA), 250 µg/ml denatured salmon sperm DNA, and 0.1% SDS for 3 h at 42°C. The filters were then hybridized overnight in the same conditions with the indicated cDNA probes in the following order: PRL receptor, LH receptor, FSH receptor, and β-actin. The filters were stripped of any remaining probe in 5 mM Tris-HCl (pH 8.0), 2 mM disodium EDTA, and 0.1 Denhardt's solution for 2 h at 65°C and were autoradiographed before hybridization with the subsequent probe.

<table><caption>TABLE 2. LH, FSH, and estradiol in lactating sows submitted to LONG (16 h/day) and SHORT (8 h/day) light duration.</caption><thead><tr><th></th><th>Group LONG</th><th>Group SHORT</th></tr></thead><tbody><tr><td><strong>LH profiles</strong></td><td></td><td></td></tr><tr><td>Mean concentration (ng/ml)</td><td>0.85 ± 0.06</td><td>0.97 ± 0.10</td></tr><tr><td>Basal concentration (ng/ml)</td><td>0.82 ± 0.04</td><td>0.95 ± 0.09</td></tr><tr><td>No. of pulses/6 h</td><td>0.40 ± 0.20</td><td>0.50 ± 0.40</td></tr><tr><td><strong>FSH</strong></td><td></td><td></td></tr><tr><td>Mean concentration (ng/ml)</td><td>2.35 ± 0.19</td><td>2.10 ± 0.06</td></tr><tr><td><strong>Estradiol-17β</strong></td><td></td><td></td></tr><tr><td>Mean concentration (pg/ml)</td><td>13.5 ± 0.5</td><td>13.3 ± 1.4</td></tr></tbody></table>

Values are mean ± SEM; n = 8.

At the end of the hybridization period, the blots were washed once in 0.1-strength SSC, 0.1% SDS, 1 mM EDTA for 10 min at room temperature; they were then washed twice in the same solution for 30 min at 60°C. Filters were exposed, with use of two intensifying screens, to Amersham hyperfilm for either 12 h (LH receptor) or 14 days (FSH and PRL receptors) at -70°C. Autoradiograms were analyzed by densitometric scanning (Biocom Image Analysis System, MRAG program from Touzart & Matignon, Vitry-sur-Seine, France). Control lung samples were used to determine the threshold of negative hybridization. Hybridization intensities (expressed in arbitrary units) were calculated as signal (dot optical density) to noise (optical density of film background), and a ratio of hybridization of the signal to actin was determined. The value of the control lung ratio was used to determine the threshold of negative hybridization and was subtracted from each determination. Control testicle (FSH receptor, LH receptor) or mammary gland (PRL receptor) samples were used to determine the threshold of positive hybridization.

### Statistical Analyses

Number of LH pulses was determined according to the iterative procedure described by Merriam and Wachter [33] with the same parameters as previously used [34].

Percentages of sows returning to estrus within 10 days after weaning were compared using the 2t test, which is derived from chi-square analysis [35]. Plasma concentrations of LH and FSH were examined by analysis of variance using the General Linear Models procedure of the SAS package [36]. For both hormones, a split-plot design was used that included light regimen, sow nested within light regimen, time, and time × light regimen as sources of variation. Ovarian weight, size of the largest follicle, and estradiol-17β concentrations were compared by Student's t-test. Receptor data and number of LH pulses were analyzed with the non-parametric test of Wilcoxon using the NPAR1WAY procedure of the SAS package. Nonparametric coefficients of correlation were calculated between variables by the method of Spearman using the FREQ procedure of SAS. Means and SEM were calculated.

## RESULTS

### Return to Estrus after Weaning

The 4 sows submitted to the SHORT light duration and checked for estrus after weaning exhibited an estrus rapidly, at 6 (n = 2) or 7 (n = 2) days. In the 5 sows checked for estrus after weaning in the LONG group, two had an estrus at 4 and 7 days, respectively, while estrus was delayed beyond 10 days in the 3 others (*p* < 0.05). Progesterone determination in these 3 sows confirmed that they had not ovulated.
<table><caption>TABLE 3. LH, FSH, and PRL ovarian receptor expression in sows submitted to LONG (16 h/day) or SHORT (8 h/day) light duration.</caption><thead><tr><th></th><th>Group LONG</th><th>Group SHORT</th></tr></thead><tbody><tr><td>Receptor expression<br>(% of specific binding)</td><td></td><td></td></tr><tr><td>R-LH</td><td>16.2 ± 1.1</td><td>14.5 ± 1.5</td></tr><tr><td>R-FSH</td><td>4.4 ± 1.0</td><td>4.2 ± 0.4</td></tr><tr><td>Receptor mRNA<br>(relative expression)</td><td></td><td></td></tr><tr><td>R-LH</td><td>1.3 ± 0.3<sup>a</sup></td><td>0.7 ± 0.5<sup>b</sup></td></tr><tr><td>R-FSH</td><td>1.6 ± 1.3</td><td>0.9 ± 0.7</td></tr><tr><td>R-PRL</td><td>3.8 ± 1.4</td><td>2.7 ± 1.4</td></tr></tbody></table>

Values are means ± SEM; n = 8.

## Ovarian Morphology and Plasma Hormones

In all the sows, CL from the previous gestation were re-gressed. In both groups, none of the superficial follicles had a diameter greater than 5.5 mm and no follicular cysts were present. Wet weight of both ovaries and diameter of the largest follicle per sow were similar in the LONG and SHORT groups (Table 1, *p* > 0.1). Serial profiles of plasma LH were highly variable, as shown in Figure 2. Pulses of LH could be identified in only 5 of 16 sows, and their number during 6 h ranged between 1 and 3. Number of LH pulses and mean and basal concentrations of LH did not differ between the two groups (Table 2). Similarly, concentrations of plasma FSH and estradiol-17β were not influenced by light duration (Table 2).

## Total Gonadal RNAs of FSH, LH, and PRL Receptors

Figure 3 displays the results obtained from a typical slot-blot experiment for each probe. This type of experiment produces quantifications of all the transcripts for each of the receptors [31, 37]. The mRNA for the LH receptor was abundant, since 12 h of autoradiography sufficed to detect its presence, while the other receptors required 14 days of autoradiographic exposure.

Levels of hybridization with the cDNA [α³²P]-labeled probe were positive in 12 of 16 sows for the LH receptors, in 4 of 16 sows for the FSH receptors, and in 11 of 16 sows for the PRL receptors. Levels of hybridization with the FSH and PRL receptor cDNA probes were similar in the SHORT and LONG groups, whereas hybridization with the LH receptor probe was significantly higher in sows submitted to the longer light duration (Table 3).

<figure><img src="image_3.png" /><figcaption>FIG. 2. Typical gonadotropin profiles of sows at Day 20 of lactation. Arrows indicate the occurrence of an LH pulse.</figcaption></figure>

Light duration had no significant influence on ovarian binding of the LH and FSH receptors (Table 3). Ovarian binding analysis was also performed on PRL receptors, but the values were low for both groups of sows (1.5 ± 0.8% vs. 1.3 ± 0.5%, LONG and SHORT groups respectively). Scatchard analysis was also performed for FSH and LH receptors on individual animals from both groups. The affinity and number of receptors varied little between groups, with no relationship to day-length treatment (Table 4).

## Hormone Concentrations

Mean concentration of plasma LH was positively correlated to the specific binding of ovarian LH and FSH receptors but not to the levels of hybridization with their cDNA probes (Table 5). Plasma FSH was positively correlated to the level of hybridization with the FSH and PRL receptor probes.

## DISCUSSION

To our knowledge, this is the first attempt to determine the influence of day length on hormonal profiles and ovarian receptors in lactating gilts, as well as its impact on the expression of ovarian FSH, LH, and PRL receptors and their mRNAs.

After exposure to a SHORT day length, a significantly

<table><caption>TABLE 4. Results obtained by Scatchard analysis for FSH and LH ovarian receptor expression in sows submitted to LONG (16 h/day) or SHORT (8 h/day) light duration.<sup>a</sup></caption><thead><tr><th rowspan="2"></th><th colspan="2">R-FSH</th><th colspan="2">R-LH</th></tr><tr><th>Group LONG</th><th>Group SHORT</th><th>Group LONG</th><th>Group SHORT</th></tr></thead><tbody><tr><td>Number of sites<br>(fmol/mg of protein)</td><td>13.7 ± 3.3 (n=3)</td><td>21.5 ± 6.9 (n=4)</td><td>11.4 ± 3.5 (n=6)</td><td>15.5 ± 1.6 (n=7)</td></tr><tr><td>K<sub>d</sub> (nM)</td><td>0.21 ± 0.03 (n=3)</td><td>0.26 ± 0.04 (n=4)</td><td>0.06 ± 0.01 (n=6)</td><td>0.07 ± 0.01 (n=7)</td></tr></tbody></table>

<footnote><sup>ab</sup>Within a row, means with different superscripts differ significantly (p < 0.05).</footnote>

<footnote><sup>a</sup>Values are means ± SEM; the number of animals studied is indicated in parentheses.</footnote>
<figure><img src="image_4.png" /><figcaption>FIG. 3. Expression of FSH, LH, and PRL receptors in ovaries of lactating sows exposed to short or long light duration. Total RNAs were isolated from all sows under both light regimens. Two, four, and eight micrograms were slot-blotted and quantified in arbitrary units. Levels of mRNA expression were analyzed using the following cDNA probes: **upper left** pFSH receptor (0.8 kb); **lower left** pLH receptor (2.2 kb); **upper right** rabbit PRL receptor (0.6 kb); and **lower right** a mouse β-actin cDNA probe, used as a control. Control testicle (FSH, LH receptors) or mammary gland (PRL receptor) samples were used as positive controls (data not shown). Lung control samples were used to determine the threshold of negative hybridization.</figcaption></figure>

<table><caption>TABLE 5. Observed correlations between mean concentrations of plasma gonadotropins and ovarian receptors in lactating sows.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Mean concentration of plasma</th></tr><tr><th>LH</th><th>FSH</th></tr></thead><tbody><tr><td>LH receptor specific binding</td><td>0.51<sup>a</sup></td><td>-0.19</td></tr><tr><td>FSH receptor specific binding</td><td>0.55<sup>a</sup></td><td>-0.14</td></tr><tr><td>PRL receptor specific binding</td><td>-0.31</td><td>0.08</td></tr><tr><td>LH receptor mRNA</td><td>-0.15</td><td>0.33</td></tr><tr><td>FSH receptor mRNA</td><td>-0.12</td><td>0.61<sup>a</sup></td></tr><tr><td>PRL receptor mRNA</td><td>-0.10</td><td>0.47<sup>a</sup></td></tr></tbody></table>

higher proportion of sows exhibited estrus shortly after weaning than in the LONG group. Although only a limited number of observations were performed, this result confirms previous observations on the influence of light duration on the return to estrus after weaning [8, 10].

Our data did not indicate differences between the two groups in the number of LH pulses or mean or basal plasma concentrations of LH. Plasma concentrations of FSH and estradiol-17β were also similar in the two groups. This lack of influence of light duration on circulating levels of gonadotro-

<footnote><sup>a</sup>Correlations with a superscript are significant (p < 0.05); n = 8.</footnote>
pin in lactating sows is in agreement with previous results obtained in lactating sows submitted to gradual variations (FSH, estradiol-17β [8]). LH pulsatility was very low in both groups of sows compared to prepubertal and cyclic females [19, 38, 39], showing that LH pulsatility was inhibited during lactation, in agreement with previous studies [40, 41].

Failure of light duration to influence binding to FSH and LH receptors or to influence dissociation constants strongly suggests that the ovarian content of FSH and LH receptors was not affected by photoperiod in both groups of sows.

Even though circulating levels of PRL are still high at 3 wk postpartum, they did not seem to interfere with gonadotropin stimulation. Plasma FSH levels were correlated with the PRL receptor mRNA. This result agrees with findings from studies using rat granulosa cells in vivo and in vitro [42] in which FSH induced functional PRL receptors. Our results suggest that the stimulation could occur via newly transcribed PRL receptors.

The existence of positive correlations between mean plasma concentrations of gonadotropins and their ovarian receptors or mRNA is in agreement with the role of gonadotropins in stimulating their own receptors [43-45]. The positive correlation between plasma FSH concentration and its cognate receptor mRNA indicates that this hormone could stimulate its own receptor mRNA. The mean concentration of plasma LH was correlated with FSH and LH receptor content, thereby maintaining an ovarian sensitivity to both gonadotropins.

As indicated in the introduction, the influence of day length on the return to estrus is most likely a multifactorial phenomenon, and individual parameters may exhibit only slight variation. Here, ovarian receptors for FSH and PRL did not differ significantly between groups, nor did the relative expression of their respective mRNAs differ. The only significant change was found in the relative LH receptor mRNA content, which was lower in lactating sows submitted to the short light duration. Although the amount of LH receptor was similar in the two groups of females, this result suggests the existence of posttranscriptional regulation mechanisms that could differ between groups. Such mechanisms of regulation at the posttranscriptional level have already been observed for the LH receptor gene [43]. For instance, in porcine granulosa cells cultivated in vitro, mRNA level is relatively high, whereas LH receptor binding is relatively low, upon stimulation by corticosteroids [43]. Several groups of investigators have shown that in the rat ovary, among the different transcripts revealed by Northern blotting only a minority could encode a full-length LH receptor [46, 47]. In the neonatal rat ovary, although RNAs that hybridized with the LH receptor probe were always present, the appearance of LH receptor binding was concomitant with the detection of full-length transcripts at 7 days of age [48]. In the rat testis, the degeneration of Leydig cells induced by ethane dimethyl sulfonate was accompanied by

disappearance of the LH receptor itself and its transcripts as observed on Northern blots, with the exception of the 1.8-kb band [49]. Thus, the possibility exists that an overabundance of LH receptor RNA relative to the receptor itself may be related to the physiological state of the ovarian follicles. As total RNAs were slot-blotted in our study, we were unable to quantify the relative abundance of the different transcripts. Nevertheless, a difference in the pattern of the transcripts of the LH receptor gene may explain the observation that the LH receptor content was similar in the LONG and SHORT groups despite a higher level of mRNA in the LONG group. Moreover, this could be correlated with a lack of follicular responsiveness after weaning in LONG females, resulting in a longer weaning-to-estrus interval.

Although day length has an overt effect on the weaning-to-estrus interval, no clear molecular difference could be observed between lactating sows exposed to contrasting photoperiods. Thus, new studies are needed in order to obtain precise information on individual follicles at the end of lactation or shortly after weaning. Moreover, it seems necessary to discriminate between LH receptor transcripts. This will open the way to future studies aimed at characterizing the molecular control of LH receptor transcript maturation in correlation with the physiological state of animals.

## ACKNOWLEDGMENTS

We acknowledge the skillful technical assistance of Lucette Belair and Anne-Marie Mounier. We are indebted to the Center for Population Research (NIH, Bethesda, MD, USA) for the gift of hCG through the courtesy of Dr. R. Canfield. We also thank Dr. Y. Combarnous (Station de Physiologie de la Reproduction INRA, 37380 Nouzilly, France) for the gifts of purified porcine LH and pure and crude preparations of FSH.

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