Abstract
Purpose
The infusion of a bacterial solution into the uterus of rats raises the progesterone (P4) concentration in serum and extends diestrus. To understand the origin of the P4, we investigated the change in the P4 concentration of seven groups of rats for 5 days after the infusion of a bacterial solution.
Methods
The rats were divided into 7 treatment groups as follows: OvxBac, AdxBac, Ovx, Adx, LapBac, Lap, and Cont. In OvxBac, rats received both ovariectomy and bacterial inoculation into their uterus. In AdxBac, rats received both adrenalectomy and bacterial inoculation into their uterus. In Ovx, rats received only ovariectomy. In Adx, rats received only adrenalectomy. In LapBac, rats received only bacterial inoculation into their uterus. In Lap, rats received only laparotomy. In Cont, rats did not receive any treatment and acted as controls.
Results
The P4 concentration in all treatment groups was higher than in Cont on day 1 (the day following operation) and day 2. In Lap and LapBac, the P4 concentration was high on day 1 (>30 ng/mL) and maintained that value until day 2. In Adx and AdxBac, the P4 concentration was average on day 1 (approximately 25 ng/mL) and increased on day 2 to a value close to that of LapBac and Lap. In OvxBac, although the P4 concentration increased slightly on day 1 and day 2, it reached 22.5 ± 7.5 ng/mL on day 4. In AdxBac and LapBac, the P4 concentration on day 4 tended to be high.
Conclusion
These results suggest that P4 is initially secreted from the ovaries and the adrenals in response to the surgical stress of laparotomy, and is later secreted from the adrenals due to the inflammatory reaction of the uterus.
Keywords: Adrenalectomy, Ovariectomy, Progesterone, Rat, Uterus
Introduction
There is a close relationship between uterine and ovarian function, e.g., the endotoxin produced in the uterus during endometritis interrupts the release of gonadotropin‐releasing hormone (GnRH) from the hypothalamus and suppresses folliculogenesis or prevents ovulation [1, 2, 3]. However, the mechanism by which the uterus influences ovarian function has not been fully elucidated to date. Kaneko et al. [4] reported that the infusion of a bacterial solution into rat uterine lumen caused endometritis and interrupted estrous cycle. The increase of progesterone (P4) concentration in serum after bacterial inoculation was thought to be one factor for ovarian quiescence, and this P4 increase may also depress gonadotropic hormone secretion. Although the ovaries and adrenal cortex are considered to be the origin of P4 in blood, it has not been clarified. To determine the origin of P4, we measured P4 in serum after ovariectomy or adrenalectomy in rats that had received bacterial inoculation into their uterus.
Materials and methods
Animals
Adult female Wistar rats weighing more than 220 g, inbred in this laboratory, were used in all experiments. Vaginal smears were taken daily and only those animals showing 3 consecutive 5‐day estrous cycles were used. They were kept in a temperature‐controlled room (22°C) under a standard lighting regimen (12 h light:12 h dark, lights off at 2000 h) and provided with food (standard laboratory rat chow) and water ad libitum. All experiments were carried out in accordance with ethical consent from Azabu University.
Treatment
We divided the rats into seven treatment groups consisting of 25 rats each: OvxBac, AdxBac, Ovx, Adx, LapBac, Lap, and Cont. In OvxBac, rats received both ovariectomy and bacterial inoculation into their uterus. In AdxBac, rats received both adrenalectomy and bacterial inoculation into their uterus. In Ovx, rats received only ovariectomy. In Adx, rats received only adrenalectomy. In LapBac, rats received only bacterial inoculation into their uterus. In Lap, rats received only laparotomy. In Cont, rats did not receive any treatment and acted as controls. Each treatment was administered during metestrus. Laparotomy, ovariectomy or adrenalectomy was performed via a midventral incision under anesthesia with a subcutaneous injection of 8 mg pentobarbital sodium (Nembutal, Abbott laboratory, Chicago, IL, USA). The bacterial solution was prepared by dissolving Staphylococcus aureus colonies growing on sheep blood agar into physiological saline and creating 2–15 × 108 colony‐forming units (CFU)/mL. This bacterial solution of 0.03 mL was injected into the uterine lumen via a 26‐gauge needle in the OvxBac, AdxBac and LapBac groups. At each treatment, 2 mg crystalline benzyl penicillin potassium dissolved in 1 mL physiological saline was administered into the abdominal cavity of all the rats; each treatment was performed between 10 and 12 a.m. during metestrus.
Hormone analysis
In each group, five rats were sacrificed every day from the day following the operation (described as day 1) to day 5 and blood samples were collected. After anesthesia as described above and laparotomy between 10 and 12 a.m., blood was collected with a 26‐gauge needle from the abdominal aorta. The blood was allowed to clot at room temperature and centrifuged, after which the serum was removed and frozen at −18°C until the hormone assay was performed. The serum concentration of P4 was analyzed. P4 concentration was measured in all samples by radioimmunoassay without extraction using a commercial kit (Diagnostic Products, Los Angeles, CA, USA). Cross‐reactivities of the anti‐P4 antibody for progesterone, 5α‐pregnane‐3‐20‐dione, 17α‐hydroxyprogesterone, 5β‐pregnan‐3‐20‐dione, 20α‐dihydroprogesterone, testosterone, 5β‐pregnane‐3α‐ol‐20‐one, androstenediol and 17β‐estradiol were 100, 9, 3.4, 3.2, 0.2, 0.1, 0.05, <0.05 and <0.05%, respectively. Intra‐ and interassay coefficients of variation were 8.8 and 9.7%, respectively, and the detection limit was 0.02 ng/mL.
Histological examination
The uteri were fixed in 10% formaldehyde at the time of blood collection, embedded in paraplast, sectioned at 4 μm, and stained with hematoxylin and eosin for histological examination.
Statistical analysis
The serum concentration of P4 was analyzed using ANOVA, followed by Fisher's protected least‐significant difference post‐hoc analysis. Values are presented as mean ± standard error of the mean. Values of P < 0.05 were considered statistically significant.
Results
The change in P4 concentration is shown in Table 1. P4 concentration in all treatment groups on day 1 was higher than that in Cont. In Lap and LapBac, P4 concentration showed a similar change irrespective of the existence of bacteria inoculation in their uterus. P4 concentration was high on day 1 (>30 ng/mL) and maintained that value until on day 2; it then decreased sharply at day 3 (<15 ng/mL) (Table 1). In Adx and AdxBac, P4 concentration showed a similar change irrespective of the existence of bacteria inoculation in their uterus. P4 concentration was average on day 1 (approximately 25 ng/mL) and increased on day 2 to a value close to that of LapBac and Lap; it then decreased sharply on day 3 (<10 ng/mL) (Table 1). In Ovx and OvxBac, P4 concentration increased slightly, but it was not as high as in Lap, LapBac, Adx or AdxBac on day 1 and day 2. However, it increased and reached 22.5 ± 7.5 ng/mL on day 4 in OvxBac, and 15.0 ± 2.3 ng/mL on day 5 in Ovx (Table 1). In LapBac and OvxBac, P4 concentration on day 5 was higher than that of the other four groups and as high as Cont at proestrus (Table 1). The P4 secretion has two phases; at first P4 is secreted from the ovaries and later it is secreted from the adrenals.
Table 1.
The change in P4 concentration in each group
| Group | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 |
|---|---|---|---|---|---|
| LapBac | 44.5 ± 17.9 | 36.8 ± 21.2 | 13.5 ± 3.2m | 8.7 ± 2.9o | 5.9 ± 1.2s |
| Lap | 32.6 ± 3.5a | 39.2 ± 12.9g | 5.5 ± 2.2 | 3.9 ± 1.0 | 5.7 ± 2.3t |
| AdxBac | 22.4 ± 3.7b | 37.8 ± 2.8h | 7.7 ± 3.9 | 2.2 ± 0.4p | 2.5 ± 0.7u |
| Adx | 26.0 ± 4.8c | 35.6 ± 6.8i | 2.4 ± 0.5n | 1.9 ± 0.7q | 3.4 ± 0.7v |
| OvxBac | 19.5 ± 3.3d | 18.6 ± 3.8j | 14.6 ± 4.9m | 22.5 ± 7.5r | 9.9 ± 2.4w |
| Ovx | 12.9 ± 2.3e | 15.1 ± 6.4k | 7.7 ± 2.6 | 2.4 ± 0.9 | 15.0 ± 2.3x |
| Cont | 8.1 ± 2.6f | 4.8 ± 1.1l | 16.3 ± 4.6m | 6.0 ± 1.5 | 11.4 ± 4.3 |
Mean ± SEM. OvxBac received ovariectomy and bacterial inoculation into their uterus; AdxBac received adrenalectomy and bacterial inoculation into their uterus; Ovx received only ovariectomy; Adx received only adrenalectomy; LapBac received only bacterial inoculation into their uterus; Lap received only laparotomy; Cont is control. In Cont, day 1 is proestrus, day 2 is estrus, day 3 is metestrus, day 4 is the first day of diestrus, day 5 is the second day of diestrus. There is a significant difference between a and d, a and e, a and f, c and e, c and f, f and b, f and d (Day 1); l and g, l and h, l and i, l and j, h and j, h and k (Day 2); n and m (Day 3); r and o, r and p, r and q, o and q (Day 4); x and s, x and t, x and u, x and v, w and u, w and v, t and u (Day 5) (P < 0.05)
In rats that received bacterial inoculation into their uterus, moderate cellular infiltration, mainly consisting of neutrophils, was observed in the stratum compactum on day 1. Crudeness of collagenous fibers and collapse of stroma structure occurred on day 2. On day 4, fibrocytic infiltration started and vacuolation, cornification, and stratification were observed in epithelial cells; these changes were also observed in epithelial cells of uterine glands. Although these changes were moderate on day 1, they gradually became evident from day3 and continued during the observation period.
Discussion
On day 1, P4 concentration rose in all groups after each treatment. In particular, it increased the most in Lap and LapBac in which both ovaries and adrenals were intact. Milenković et al. [5] reported that P4 concentration in blood increased as a result of dock stress in rats and this P4 increase was higher in the animals in which both ovaries and adrenals were intact than in the animals in which either ovaries or adrenals were excised. The result in our study was consistent with Milenković's report [5]. The large increase in P4 concentration on day 1 in Lap and LapBac might be due to the combination of P4 that originated from both the ovaries and the adrenals. Similar to Lap and LapBac, the P4 concentration increased significantly in Adx and AdxBac in which only the ovaries were intact, and this P4 increase must have originated from the ovaries. The extent of the P4 increase was smallest in Ovx and OvxBac in which only the adrenals were intact, and this P4 increase must have originated from the adrenals. These results indicate that although P4 was secreted from both the ovaries and adrenals on day 1, the ovaries secreted much more P4 than the adrenals. On day 2, the P4 concentration in Lap, LapBac, Adx and AdxBac was high and similar in all four groups. However, the P4 concentration in Ovx and OvxBac, in which the P4 originated from only the adrenals, did not increase from day 1 level, and was not as high. Therefore, it was thought that most of the P4 in Lap, LapBac, Adx and AdxBac on day 2 originated from the ovaries, and then decreased rapidly on day 3. On day 4, the P4 concentration in OvxBac was the highest and it must have originated from the adrenals because only the adrenals were intact in OvxBac.
Irrespective of whether the bacterial solution was inoculated or not, P4 concentration increased on day 1 and day 2 in all treatment groups; therefore, the increase of P4 concentration might not be due to bacteria in the uterus. In the groups in which the ovaries were intact (Adx, AdxBac, Lap and LapBac), the P4 concentration increased mostly on day 1 and day 2. Surgical stress affects prolactin (PRL) secretion via the opioid system, and the PRL stimulates P4 secretion from the corpus luteum [5, 6]. Therefore, there was a possibility that the increase of P4 concentration on day 1 and day 2 in Lap, LapBac, Adx and AdxBac was induced by PRL.
In the groups in which only the adrenals were intact (Ovx and OvxBac), P4 concentration increased slightly on day 1 and day 2. Acute stress leads to the rapid release of corticotrophin releasing hormone (CRH) and vasopressin (VP) into the portal blood system of the pituitary [7, 8]. CRH and VP synergistically act to stimulate the secretion of adrenocorticotropic hormone (ACTH) from the anterior pituitary [9]. Plasma ACTH stimulates the synthesis and secretion of glucocorticoids from the adrenal gland, and glucocorticoid and ACTH itself increase P4 secretion from the adrenals [10, 11]. Furthermore, Resko [12] reported that surgical stress on ovariectomized rats caused a release of P4 from the adrenals. These reports make us confident that the P4 on day 1 and day 2 in Ovx and OvxBac originated from the adrenals.
Although the P4 concentration in the other four groups on day 3 was lower than that of Cont, P4 concentration in LapBac and OvxBac was as high as that of Cont. Both LapBac and OvxBac had adrenals and received bacterial inoculation. Furthermore, in OvxBac, the P4 concentration increased on day 4 even if there had not been a large increase on day 1 and day 2; also the second level of P4 on day 4 was marked in LapBac. These results suggest that bacteria in the uterus stimulate P4 secretion from the adrenals. As a result, inflammatory cytokine‐like interleukin (IL)‐1 elevates ACTH [13, 14], and stimulates P4 secretion from the adrenals [15]. Xiao et al. [16] also reported that IL‐1 stimulated P4 secretion from the adrenals in the ovariectomized rhesus monkey; therefore, the P4 concentration rise observed in OvxBac on day 4 might have been due to these mechanisms.
Kaneko et al. [4] reported that bacterial inoculation into the uterus of rats prolonged anestrus to 9.3 days from the original 2 days, and they concluded that the P4, which was increased in blood after bacterial inoculation, was the main reason for the extension of anestrus. Our results support their theory since the P4 concentration increased in all the treatment groups. The negative feedback effect of P4 to the hypothalamus may cause the extension of anestrus. Furthermore, the results of this study show that the origin of P4 is in both the ovaries and the adrenals. This mechanism maintains an elevated P4 level, and extends anestrus for a long period. It had been reported that PRL and CRH depressed the secretion of GnRH [17, 18, 19]; however, we need to measure PRL, CRH, ACTH and cytokine to verify this mechanism.
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