The present study aimed to determine if gossypol interferes with ovarian follicles in rats. Twenty-four female Wistar rats were assigned to two equal groups: one control group and the other dosed with gossypol (25 mg/kg/day, subcutaneously) for 15 days. Ovarian follicles were histologically classified according to the stage of development and as normal or atretic. Gossypol treatment reduced the length of estrous with an increase in the duration of the diestrus phase. This compound was responsible for reduced serum levels of T4 and progesterone. Treatment with gossypol was responsible for a significant reduction in the number of normal ovarian follicles and a significant increase in the number of atretic follicles, both in all stages of development. Thus, treatment of rats with gossypol was responsible for reduction in the number of viable follicles and changes in hormone levels that resulted in interference of the estrous cycle.
Gossypol (2,2-bi(8-formyl-1,6,7-trihydroxy-5-isopropyl-3-methylnaphthalene)) is a polyphenolic compound produced by pigment glands in roots, stems, leaves, seeds, and flower buds of cotton (
The male reproductive toxicity of gossypol has been reported in many studies [
Even though gossypol has well-known toxic effects in the male reproductive system, there are few studies on its effects on female reproduction. Female exposure to gossypol has been associated with interference of the estrous cycle, pregnancy, and early embryonic development [
Female Wistar rats, 60 to 70 days old, weighing approximately 120 g each, from the Animal Sciences Department, UFERSA, Mossoró, RN, Brazil, were used. During the entire study period, the rats were housed in plastic cages under controlled environmental conditions with a 12 h light/dark cycle and at a temperature of
The estrous cycle of the rats was monitored twice a day by vaginal smear for seven consecutive days; only rats that exhibited regular estrous cycles were used in the experiment. Twenty-four female rats were randomly and equally distributed into two groups: a control group (injected with saline solution subcutaneously) and a group dosed with gossypol (25 mg/kg/day subcutaneously) for 15 consecutive days. Rats were dosed with (+/−) gossypol acetic acid (Fluka, G4382) with purity about 95%.
Vaginal smears were carried out on each animal every day between 7:00 and 9:00 a.m. and again between 5:00 and 6:00 p.m. The smears were collected by lavage with 20
On the day after the last dosing, all rats were deeply anesthetized by intraperitoneal injection of xylazine (5 mg/kg) and ketamine (60 mg/kg) to collect the ovaries, uterus, thyroid glands, and blood samples from the vena cava. Formalin-fixed, paraffin-embedded tissue samples were sliced into 5
Frozen sera were used for the determination of the serum levels of estradiol, progesterone, luteinizing hormone (LH), thyroxine (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH). Hormone levels were analyzed by an ELISA automated analyzer (Elisys Uno, Human, Wiesbaden, Germany) with specific kits (RPC Diagnostic Systems, Nizhny Novgorod, Russia).
The ovaries from all rats were collected and fixed in 10% buffered formalin. Five-micron thick paraffin-embedded sections were collected at 60
Ovaries from control rats showing normal (a) primordial, (b) primary, (c) secondary (H&E, bar = 25
Follicles were also classified as normal or atretic. Normal follicles presented a regular shape and well-organized granulosa cells, without signs of atresia. Atretic follicles were characterized by retracted oocytes, a pyknotic nucleus, discontinued basement membrane, and disorganized granulosa cells [
The obtained data were statistically analyzed using R software (version 3.0.0). Data normality was evaluated using a Shapiro-Wilk test and the homogeneity of variance was evaluated using an
No clinical signs of poisoning were observed in any of the rats. There was no significant difference in body weight between the groups on the first and last days of the experiment, but body weight gain was significantly (
Body weight, body weight gain (in grams), and length (in hours) of the estrous cycle phases of female rats dosed with gossypol (25 mg/kg/day) or 0.9% saline (control group) subcutaneously for 15 consecutive days.
Control ( |
Gossypol ( |
|
|
---|---|---|---|
Body weight (g) | |||
Day 0 |
|
|
n.s. |
Day 15 |
|
|
n.s. |
Body weight gain (g) |
|
|
0.0006792 |
Diestrus (h) |
|
|
0.01875 |
Metestrus (h) |
|
|
n.s. |
Estrus (h) |
|
|
n.s. |
Proestrus (h) |
|
|
n.s. |
n.s.: not significant (
At necropsy, hydrometra was observed in five of the rats dosed with gossypol and in one rat from the control group. No histological lesions were found in the thyroid glands or the uterus from any of the rats.
Our results also verified that gossypol was responsible for hormonal interference, characterized by significantly (
Serum concentrations of estradiol, progesterone, luteinizing hormone (LH), thyroxine (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH) in female rats dosed with gossypol (25 mg/kg/day) or 0.9% saline (control group) subcutaneously for 15 consecutive days.
Control ( |
Gossypol ( |
|
|
---|---|---|---|
T4 (ng/dL) |
|
|
0.01446 |
T3 (pg/mL) |
|
|
n.s. |
T3/T4 ratio |
|
|
0.02635 |
TSH ( |
|
|
n.s. |
Progesterone (ng/mL) |
|
|
0.01317 |
LH (mIU/mL) |
|
|
n.s. |
Estradiol (pg/mL) |
|
|
n.s. |
n.s.: not significant (
Additionally, treatment with gossypol was responsible for a significant (
Ovarian follicle populations in female rats dosed with gossypol (25 mg/kg/day) or 0.9% saline (control group) subcutaneously for 15 consecutive days.
Follicles | Control ( |
Gossypol ( |
|
---|---|---|---|
Number of normal follicles | |||
Primordial |
|
|
<0.0001 |
Primary |
|
|
0.0008635 |
Secondary |
|
|
0.003426 |
Antral |
|
|
<0.0001 |
Total |
|
|
|
|
|||
Number of atretic follicles | |||
Primordial |
|
|
0.001489 |
Primary |
|
|
<0.0001 |
Secondary |
|
|
0.00023 |
Antral |
|
|
<0.0001 |
Total |
|
|
|
|
|||
Proportion of follicles (in %) | |||
Normal |
|
|
<0.0001 |
Atretic |
|
|
<0.0001 |
|
|||
Number of total follicles | |||
Total |
|
|
n.s. |
n.s.: not significant (
(a) Ovaries from gossypol treated rats showing atretic primordial, primary, and secondary follicles (H&E, bar = 100
We found that gossypol treatment resulted in increase in the length of the diestrus and metestrus stages (about 116% and 350% of controls, resp.) and reduction in the length of the proestrus and estrus stages (both about 81% of controls). Interferences on estrous cycle were observed by others [
Our results verified that gossypol is responsible for hormonal interference, as evidenced by reduced serum levels of progesterone and T4 and an increased T3/T4 ratio. Reduced serum levels of progesterone were reported in female rats that received 25 mg/kg gossypol acetic acid via intramuscular injection for 3 to 5 days [
Gossypol is also an antithyroidal compound [
The treatment with gossypol was also responsible for both a significant reduction in the number of normal follicles and a significant increase in the number of atretic follicles in all stages of development. On average, the ovaries of the rats in the control group had 83.3% normal follicles and 16.7% atretic follicles, whereas the rats treated with gossypol had 36.6% normal follicles and 63.4% atretic follicles. All types of normal follicle cell types are reduced about a third and the atretic follicle cell types are increased about three times. The ovarian weight was decreased in rats dosed with 5 or 10 mg gossypol/kg/day subcutaneously for 20 days [
In female mammals, the number of ovarian primordial follicles is fixed at the time of birth. The primordial follicles grow into primary, secondary, and antral follicles in a continuous and nonreversible process. The damage to primary, secondary, and antral follicles may lead to temporary infertility when the primordial follicles are not affected. On the other hand, the injury to primordial follicles may result in permanent infertility because of the eventual depletion of the pool of these follicles [
It was observed that gossypol inhibited steroidogenic activity and redox status and stimulated the vascular endothelial growth factor production in an
An additional interesting observation made at necropsy was the occurrence of hydrometra in five rats from the gossypol treated group and one rat from the control group. High incidence of infertility and atrophy of the uterus was observed in women exposed to gossypol through the ingestion of cottonseed oil in China [
In conclusion, treatment with gossypol was responsible for interference of the estrous cycle in the rats. This interference involved a reduction in the number of viable follicles as well as hormonal changes, which might negatively impact female reproduction. The overall results from the present study show the relevance of ovarian follicle classification and quantification in the determination of estrous cycling. This is pertinent for future studies that aim to determine the effects of toxic substances on ovarian metabolism.
The authors declared no potential conflict of interests with respect to the research, authorship, and/or publication of this paper.
Michelly Fernandes de Macedo was supported by postdoctoral fellowship from CAPES-PNPD (project no. 23038.007325/2011-52). This research has been supported by the grant of the CNPq (project no. 474432/2012-2) and support for language editing of the Pró-Reitoria de Pesquisa of the Universidade Federal de Minas Gerais (Edital PRP-UFMG 03/2013).