Endocrine DOI 10.1007/s12020-013-0092-y

ORIGINAL ARTICLE

Roles of thyroid hormones in follicular development in the ovary of neonatal and immature rats Jaafar Sulieman Fedail • Kaizhi Zheng • Quanwei Wei • Lingfa Kong • Fangxiong Shi

Received: 21 July 2013 / Accepted: 16 October 2013 Ó Springer Science+Business Media New York 2013

Abstract Thyroid hormones (TH) play a critical role in ovarian follicular development, maturation and the maintenance of various endocrine functions. However, whether TH can affect ovarian follicular development in neonatal and immature rats remains unclear. Therefore, the aim of the present study was to elucidate the effect of TH on ovarian follicular development in neonatal and immature rats. Thirty female post-lactation mothers of Sprague– Dawley rat pups were randomly divided into three groups: control, hyperthyroid (hyper), and hypothyroid (hypo). On postnatal days (PND) 10 and 21, body weights, serum hormones, ovarian histologic changes, and immunohistochemistry of thyroid hormone receptor alpha 1 (TRa1) and nitric oxide synthase types (NOS), and NOS activities, were determined. The data showed that body weights significantly decreased in both hyper and hypo groups compared with the control group (P \ 0.05). In addition, the hyper group had increased serum concentrations of T3, T4, and E2; whereas the hypo group manifested reduced serum concentrations of T3, T4, and E2 on PND 10 and 21. The hyper and hypo groups showed significantly reduced total number of primordial, primary and secondary follicles on PND 10 and 21 compared with the control group (P \ 0.05). Similarly, antral follicle numbers in the hyper and hypo groups were significantly decreased on PND 21 compared with the control group (P \ 0.05). Immunostaining indicated that TRa1 and NOS were expressed in ovarian surface epithelium and oocytes of growing and antral follicles, with strong staining of the granulosa and

J. S. Fedail  K. Zheng  Q. Wei  L. Kong  F. Shi (&) Laboratory of Animal Reproduction, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China e-mail: [email protected]

theca cells of follicles. NOS activities were significantly augmented in the hyper, but diminished in the hypo groups on PND 10 and 21. In summary, our findings suggest that TH play important roles in ovarian functions and in the regulation of NOS activity. Our results also indicate that a relationship exists between the TH and NO signaling pathways during the process of ovarian follicular development in neonatal and immature rats. Keywords Nitric oxide synthase (NOS)  Ovary  Rat  Thyroid hormone (TH)

Introduction Thyroxine (T4) is the major form of thyroid hormone (TH) found in the blood, and is secreted by follicular cells of the thyroid gland [1]; while triiodothyronine (T3) is the predominant active form of TH. In tissues, T4 is converted to the considerably more active T3 via the action of deiodinase [2]. The action of THs is mediated through binding to their specific receptors which are called thyroid hormone receptors (TRs) [3], and these are widely expressed in different tissues and organs [4]. Thyroid hormone nuclear receptors (TRs) mediate the biologic activities of T3 via transcriptional regulation [5]. In addition, two kinds of TR genes (TRa1 and 2, and TRb 1 and 2) encode the T3binding receptors [6]. In the ovary and testis of goldfish, down-regulation of TR isoforms by THs suggests that TRs are involved in the control of animal reproduction [7]. Recently, TRs were reported to be present in human ovarian surface epithelium and to act on ovarian follicles, and showing some slight localization in granulosa cells of ovarian follicles [8]. It has been reported that THs regulate a variety of biological processes including growth, cellular

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oxygen consumption, metabolism, embryonic development, tissue differentiation, and maturation [9]. In mammals, down-regulation of THs reduced fertility and decreased follicle number [10–12]. During the neonatal period, an imbalance of THs reduces the number of primordial, growing and antral follicles in mice [12]. Previous studies showed that THs down-regulated growth hormone mRNA expression in the rat ovary, and attenuated growth rate, and bone mineral density in postnatal life and adulthood [13]. Nitric oxide (NO) is a reactive free radical molecule known to exert a variety of physiological activities, particularly those involved in follicular formation and development [14, 15]. NO is a reactive species synthesized from Larginine by NO synthase (NOS), and is subdivided into neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) forms [16]. In vitro studies demonstrated that iNOS immunoreactivity was localized to theca and granulosa cells, whereas, nNOS and eNOS expressions were marked in the granulosa, theca, and cumulus cells of buffalo ovaries [17]. Expression of nNOS was observed in the organs of the reproductive system in many animals including neonatal rats [18]. Expressions of NOS subtypes were also detected in the immature rat ovary [19]. The ovarian total NOS, eNOS, and iNOS activities were increased during fetal periods compared to postnatal periods in the ovary of pig [20]; in addition, the NOS activity and NO concentration were induced at neonatal days 7 and 10 compared with others days in newborn rats [18]. NOS were also found in the ovary of fetal, neonatal, and immature pigs [20]. During quail ovarian folliculogenesis [21], it was established that NO is located in several ovarian compartments, indicating its role in steroidogenesis, follicular development, atresia, ovulation, and luteal formation [22–24]. However, the effect of TH on ovarian follicular development and NOS activity has not been investigated in neonatal rats. In the present study, we hypothesized that the induction of both hyperthyroidism and hypothyroidism in rats might alter plasma TH levels, and affect follicle numbers, as well as NOS activities. Moreover, we expected that dysfunction of the thyroid gland would modify TRa1 and nNOS localization and expressions in the rat ovary. The aim of this work is to evaluate a possible role of TH on ovarian follicular development involving the NOS signaling pathway in neonatal and immature rats.

Methods Animals Adult female pregnant rats of the Sprague–Dawley strain were obtained from the Experiment Animal Center of

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Nanjing Medical University, China. The rats were kept in an animal room with controlled temperature (21–22 °C) and lighting (12 h light, 12 h dark), and humidity at 65–70 %. Rats were fed with standard balanced rat pellets and drinking water was made available ad libitum. Experimental design Treatment was started 1 day postpartum and stopped at day 21 of weaning for the neonates. Rats were divided into three groups as follows: Controls, mothers were provided with normal drinking water and their pups were used as controls; Hypo, mother rats were administered 0.05 % 6-propyl-2-thiouracil (PTU, Sigma, and St. Louis, MO, USA) dissolved in drinking water until the end of the experiment and their pups were rendered hypothyroid as they received the drug lactation ally; and Hyper L-thyroxine (T4; Sigma Chemical Company, Sigma, and St. Louis, MO, USA) was dissolved in 0.1 mm NaOH (sodium hydroxide) solution and diluted in physiological saline; to be administered to mother rats by daily subcutaneous injections of 20 lg/100 g body weight in the same period as the PTU treatment. Tissue preparation Animals were killed after 10 and 21 days of treatment. Blood samples were collected and centrifuged at 4,0009g for 10 min to retrieve serum and then stored at -80 °C. Body weights of rats in hyper, hypo, and control groups were recorded at the end of the study period. Immediately after sacrifice, ovarian samples were collected and weighed on PNDs 10 and 21. The fresh ovaries were washed thrice with ice-cold Hank’s solution. One ovary was fixed in 40 g/L paraformaldehyde at room temperature for 24 h and then kept in 70 % alcohol for histological and immunohistochemical examination. The experimental protocols involving rats were approved in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institutional Animal Care and Use Committee of Nanjing Agricultural University, China. Radioimmunoassay (RIA) for triiodothyronine (T3), thyroxine (T4), and 17b-estradiol (E2) Serum was used to determine concentrations of T3, T4, and E2 using commercial RIA kits (Shanghai University of Traditional Chinese Medicine, China), in the General Hospital of the Nanjing Military Command, China. The sensitivity of the T3, T4, and E2 determinations was 0.2 ng/mL, 5 ng/mL, and 5 pg/mL, respectively. The intraassay coefficients of variation were \10 % and inter-assay coefficients of variation were \15 % for T3, T4, and E2.

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Histologic examination To evaluate the ovarian development of neonatal rats, ovaries kept in 70 % alcohol were dehydrated through increasing alcohol concentrations, and embedded in paraffin. The slides were stained with hematoxylin and eosin (H&E) and we observed any histopathology changes under a light microscope (Nikon Tokyo, Japan). The number of individual follicles was determined by examining five sections (the middle cross-section and four other sections) per ovary. Every section was at least 10 lm away from another section. The stages of follicles (primordial, primary, secondary, and antral) were divided based on classification criteria derived mainly from our previous laboratory studies [25, 26].

Fig. 1 Effects of thyroid hormones on body weight in rats. Values are expressed as the mean ± SEM; n = 15 animals/group. Different superscript letters indicate significant differences among groups (P \ 0.05)

Immunohistochemistry After fixation, the ovaries from the three groups were embedded in paraffin, and then 5-lm sections were cut and mounted on slides. The sections were then processed for immunohistochemical analysis using polyclonal antibodies raised in rabbits against nNOS obtained from Boster Biological Technology (Wuhan, China) and TRa1 from Abcam (Cambridge, MA, USA). The sections were incubated at room temperature overnight with polyclonal rabbit immunoaffinity-purified antisera directed against TRa1 (diluted 1:150), nNOS (1:100), iNOS (1:100), and eNOS (1:100). The immunoreactivity assay of specific protein was visualized by the Elite ABC kit (Bio Genex, San Ramon, CA, USA) and reaction with 0.05 % 3,30 diaminobenzidine tetrachloride (DAB; Sigma Chemical Co.) in 10 mmol/L phosphate-buffered saline (PBS) containing 0.01 % H2O2 for 2 min. The negative control was the use of normal rabbit serum instead of primary antibody (Boster Biological Technology). Three independent observers were asked to examine the pictures and assessed the intensity of staining using the following scale: -, no staining detected; ?, weak; ??, moderate; ???, strong staining [18, 22]. All observers evaluated all slides, and observations outside the 5–95 % confidence interval of the remaining observations of the treatment group were considered to be outlying data and were excluded from analysis. Relative levels of immunostaining were evaluated and repeated at least four times. The results represented consistently observed patterns of immunohistochemical staining [27]. Measurement of NOS activity The total NOS, iNOS, and eNOS activities were measured using a commercial reagent (Jiancheng Bioengineering Institute, Nanjing, China). Briefly, NOS activity was

determined by measuring the release of lactate NO generated via a five-electron oxidation of terminal guanidinium nitrogen of L-arginine by NOS [28]. Then, NO was bound to the nucleophilic materials and generated a colored compound. Thereafter, the reaction was terminated with citric acid. The optical density (OD) was measured at 530 nm using a SynergyTM 2 Multi-function Microplate Reader (Bio-Tek Instruments Inc., Winooski, Vermont, USA). The procedures were performed strictly according to the manufacturer’s protocols. Statistical analysis Continuous variables were expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using a commercially available program, the Statistical Package for the Social Sciences (SPSS Version 13.0; Chicago, IL, USA). Comparisons among groups were performed using one- and two-way analysis of variance (ANOVA) with Tukeys’ test for multiple comparisons P \ 0.05 was considered to be statistically significant.

Results Body weights of control, and induced hyperthyroid and hypothyroid rats Body weights of neonatal rats of control, hyper, and hypo treated rats are illustrated in Fig. 1. In the hyper group, body weights were significantly reduced on PND 10 and 21 (P \ 0.05 and P \ 0.01), compared with control. However, a very dramatic reduction in body weights was observed in the hypo rats on PND 10 and 21 (P \ 0.001) compared with controls.

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Endocrine Fig. 2 Effects of thyroid hormones on serum concentrations of T3, T4, and estradiol in rats. The serum concentrations of T3 (a), T4 (b), and E2 (c) were measured on days 10 and 21. Each bar represented the mean ± SEM n = 15 animals/group. Different superscript letters indicate significant differences among groups (P \ 0.05)

Fig. 3 Representative photographs of histologic morphology in hypothyroid, hyperthyroid and control ovaries on PND 10 and 21. Control rats showed fewer numbers of atretic follicles compared to hypo- and hyperthyroid; the latter two groups also showed augmented

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numbers of atretic follicles and unhealthy developing follicles. Se secondary follicle, An antral follicle, E atretic follicle, GC granulosa cells, TC theca cells, Oo oocyte. Scale bar, 50 lm

Endocrine Table 1 differential follicular counts in the ovary of control, hyperthyroid, or hypothyroid rats Groups

Age

Follicle type Primordial

Control

Primary

Secondary

Antral

Atretic

–

–

10

896 ± 28.75

319 ± 5.3

144.3 ± 2.2

21

443 ± 3.51

129.7 ± 3.7

55.7 ± 1.5

16 ± 0.56

Hyper

10

818 ± 1.53*

303.7 ± 3.18*

138 ± 1.5*

–

–

21

431 ± 4.16*

12 ± 1.16**

56 ± 2.1**

Hypo

10

764.3 ± 25.67**

21

393.7 ± 0.9**

120 ± 0.58* 308.3 ± 0.88 114 ± 2.31**

46 ± 1.15** 131.7 ± 0.9** 37 ± 2.6**

–

9 ± 0.58

– 8 ± 0.58**

67 ± 1.53**

Values are expressed as mean ± SEM of fifteen animals. Means in the same column were significantly different (* P \ 0.05; ** P \ 0.01)

Serum hormone concentrations in control, hyper, and hypo rats We quantified total T3, T4, and E2 levels in serum for control, hyper, and hypo groups from different aged rats, and these data are shown in Fig. 2. Total T3, T4, and E2 concentrations were augmented in the hyper group compared with control (Fig. 2a–c). In contrast, the concentrations of T3, T4, and E2 were significantly diminished on PND 10 and 21 in the hypo rats compared with controls (Fig. 2a–c). Ovarian histologic and morphometric analyses Ovarian histologic sections of hyper and hypo rats on PND 10 and 21 showed significantly reduced numbers of healthy follicles (primordial, primary, secondary); and antral follicle number was considerably less than in control rats. In a similar fashion, the number of atretic follicles was significantly higher in both experimental groups on PND 21 compared with control rats (Fig. 3; Table 1). Immunohistochemical staining of TRa 1 and NOS isoforms in the ovary of neonatal rats

observed in the granulosa and theca cells at all stages of follicular development (relative levels of TRa 1 immunostaining are shown in Tables 2 and 3). In order to investigate the localizations of NOS subtypes in the ovary during follicular development in neonatal and immature rats, immunohistochemical staining of three NOS isoforms was performed on sectioned rat ovaries (Figs. 4, 5). nNOS expression in hypo, hyper, and control groups on PND 10 and 21 was strong in the oocytes of primordial, primary, and secondary follicles, and in the granulosa cells of all follicles. Similarly, we observed staining in oocytes and granulosa cells of antral follicles at PND 21. Slight staining was also observed in the theca cells of all follicles in the hypo group. Positive expression of iNOS was detected in the oocytes and granulosa cells of primordial, growing, and antral follicles at PND 10 and 21, and iNOS staining was also found in the theca cells of antral follicles (Figs. 4, 5). eNOS immunostaining was marked in the oocytes of primordial, primary, secondary, and antral follicles (Figs. 4, 5). In addition, immunolocalization of eNOS was attenuated in granulosa and theca cells of secondary and antral follicles (relative levels of nNOS immunostaining are shown in Tables 2 and 3). Measurement of NOS activity

We evaluated the immunohistochemical staining pattern and localization of TRa 1 and NOS isoforms on PND 10 and 21 in the rat ovary using contiguous ovarian sections. In the ovary, a positive reaction was observed as brown staining. Negative controls lacking primary antibody remained unstained (Figs. 4, 5). We observed that the staining patterns were significantly different for TRa 1 and NOS isoforms between control and treated groups. The presence of TRa 1 was expressed in all follicles of rat ovaries (Figs. 4, 5). However, compared with controls, the hyper and hypo groups on PND 10 and 21 showed intense TRa 1 staining in the oocytes of follicles at the earliest stages of development, including growing and antral follicles; and marked TRa 1 staining was also

Total NOS activity was greatly enhanced in the hyper group on PND 10 (P \ 0.05) and 21(P \ 0.001), compared with controls (Fig. 6). Similarly, in the hyper group, iNOS was increased significantly compared with controls (P \ 0.05). In contrast, total NOS activity was significantly reduced in the hypo group during the neonatal period compared with controls on PND 10 and 21 (P \ 0.05). iNOS activity was also reduced on PND 10 and 21 (P \ 0.01), compared with controls. Finally, eNOS activity showed no differences among the three groups during the postnatal period; but on PND 10 eNOS activity significantly increased (P \ 0.01) in the hyper group compared with controls (Fig. 6).

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Fig. 4 Immunohistochemical staining of TRa and NOS isoforms in the ovaries of neonatal rats on PND 10. The rat was treated with T4 (hyperthyroid), PTU (hypothyroid), or drinking water (control). On PND 10, TRa 1 immunoreactivity was significantly localized to the oocytes of primordial, primary, and secondary follicles; and was found in the granulosa and theca cells of all follicles (a, b, and c). nNOS (e, f, and g), iNOS (i, j and k), and eNOS (m, n, and

o) immunoreactivity was localized to the oocytes of primordial, primary, and secondary follicles, and was also located in granulosa and theca cells of primary and secondary follicles. Negative controls lacking primary antibody remained unstained (d, h, l, and p). Pm primordial follicle, pr primary follicle, Se secondary follicle, Oo oocyte GC granulosa cells, TC theca cells. Scale bar, 50 lm

Discussion

demonstrated an effect of thyroid status on regulation of NOS activity in the ovary of newborn rats. In the current study, we examined the effects of both hyper- and hypothyroid states on body weights on PND 10 and 21. Both hyper and hypo groups showed reduced body weights. These findings are consistent with previous reports of decreased body weights in hyper- and hypothyroid rats

In the present study, we developed a greater understanding of the role(s) that TH play in rat ovarian follicular development. To the best of our knowledge, this is the first report to determine the localization and expression of TRa1 in the rat ovary before puberty. In addition, we

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Fig. 5 Immunohistochemical staining of TRa and NOS isoforms in the ovaries of neonatal rats on PND 21. The rat was treated T4 (hyperthyroid), PTU (hypothyroid), or drinking water (control). On PND 21, TRa 1 immunoreactivity was significantly localized to the oocytes of primordial, primary, secondary, and antral follicles, and was found in the granulosa and theca cells of all follicles (a, b, and c). The nNOS (e, f, and g), iNOS (i, j, and k)) and eNOS (m, n, and

o) immunoreactivity was localized to the oocytes of primordial, primary, secondary, and antral follicles: and was found in granulosa and theca cells of primary and secondary follicles. Negative controls lacking primary antibody remained unstained (d, h, l, and p). Pm primordial follicle, pr primary follicle, Se secondary follicle, Oo oocyte, GC granulosa cells, TC theca cells. Scale bar, 50 lm

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Endocrine Table 2 Relative levels of TRa 1 and NOS isoform in the rat ovary during follicular development before puberty Follicular development

Staining intensity PND 10 Hypo TRa1

Hyper

Control

nNOS

iNOS

eNOS

TRa1

nNOS

iNOS

eNOS

TRa1

nNOS

iNOS

eNOS

Oocyte Primordial

?

??

?

?

??

??

??

?

?

??

???

?

Primary

?

??

??

?

??

??

?

?

?

??

???

?

Secondary

?

??

???

??

?

???

???

??

?

???

??

?

Antral

?

??

???

??

?

???

??

??

?

???

??

?

Primordial

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

Primary

??

?

??

?

?

??

??

?

??

?

?

??

Secondary

???

??

??

?

???

??

???

?

??

??

?

??

Antral

??

??

??

?

??

??

??

?

??

??

?

??

Secondary

?

?

?

?

?

?

?

?

?

?

?

?

Antral

?

?

?

?

?

?

?

?

?

?

?

?

Granulosa cells

Theca cells

The intensity of staining is indicated by TRa 1; Thyroid hormone receptor, nNOs; neuronal nitric oxide synthase, iNOS; inducible nitric oxide synthase, eNOS endothelial nitric oxide synthase -: no staining detected; ?: weak; ??: moderate; ???: strong; NA not available; PD postnatal day

Table 3 Relative levels of TRa 1 and NOS isoform in the rat ovary during follicular development before puberty Follicular development

Staining intensity PND 21 Hypo

Hyper

Control

TRa1

nNOS

iNOS

eNOS

TRa1

nNOS

iNOS

eNOS

TRa1

nNOS

iNOS

eNOS

Primordial

??

?

??

?

?

?

??

?

??

??

?

?

Primary

??

???

??

?

??

??

??

?

??

???

??

?

Secondary

?

???

???

??

?

??

???

??

?

???

??

??

Antral

?

???

??

??

?

??

???

??

??

???

???

???

Primordial

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

Primary

??

???

?

?

??

?

?

??

??

?

??

??

Secondary

??

???

??

???

???

???

?

??

??

??

???

???

Antral

??

???

???

??

???

??

??

??

??

??

??

??

Secondary

?

?

?

?

?

?

?

?

?

?

?

?

Antral

?

?

?

?

??

?

?

?

??

?

?

?

Oocyte

Granulosa cells

Theca cells

The intensity of staining is indicated by TRa 1; Thyroid hormone receptor, nNOs; neuronal nitric oxide synthase, iNOS; inducible nitric oxide synthase, eNOS endothelial nitric oxide synthase -: no staining detected; ?: weak; ??: moderate; ???: strong; NA not available; PD postnatal day

during fetal and neonatal periods [13, 29, 30]. PTU-induced postnatal hypothyroidism also delayed growth in the female rat [31]. Neonatal hyperthyroid rats manifested inhibited

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regulation of pituitary secretion and showed reduced body weights [32]. In addition, total serum concentrations of T3 and T4 were lower in hypothyroid rats, whereas they were

Endocrine

Fig. 6 Effects of TH on nitric oxide synthase (NOS) activities if ovaries during follicular development on PND 10 and 21. There were changes in total NOS activity, iNOS activity, and no changes in eNOS

activity. Values are Mean ± SEM, n = 15 animals/group. Different superscript letters indicate significant differences among groups (P \ 0.05)

increased in hyperthyroid rats on PND 10 and 21. Our results are in accordance with previous studies in rats showing that postnatal hyperthyroid rats had elevated serum T3 and T4 levels postnatal, while neonatal hypothyroid rats showed decreased concentrations [32, 33]. One study suggested that hypothyroidism inhibits the first ovulation in eCG-primed immature female rats and that the blockage of ovulation is primarily mediated through the inhibition of the preovulatory LH surge from the pituitary [34]. Several studies in prenatal or postnatal hypothyroid rats reported disturbances of the gonadal axis at the hypothalamic-pituitary level, and decreased estradiol and increased progesterone sufficient to inhibit ovarian follicular development in rats [35]. Herein we reported that hyperthyroidism increased serum E2 concentrations, while in hypothyroid neonatal rats they were decreased. Previous studies have shown that TH deficiency modulates gonadal function neonatally via neuroendocrine mediation, and that follicular development in rats can thereby be drastically altered [36]. It was previously shown that short-term hypothyroid states augmented circulating prolactin and estradiol, and can influence ovarian follicle development in rats [37]. T4 administration to immature hypothyroid rats increased folliculogenesis and estradiol secretion [38]. Another study indicated that the levels of FSH and estrogen were significantly increased in thyrotoxicosis women [39]. Results from an in vitro study indicated that THs acted directly on the ovary, thereby increasing FSH-mediated production of progesterone and estradiol by granulosa cells [40]. Conversely, TH triggered variations in sex hormonebinding globulin (SHBG), prolactin, gonadotropin-releasing hormone, and serum steroid levels [41]. Our findings showed that the total number of follicular counts in both hyper and hypo rats during postnatal life decreased. Our study was similar to a previous report showing that the homeostatic deregulation of hyperthyroidism reduced

the number of primordial, growing, and antral follicles in mice [12]. It was reported that prepubertal hypothyroid rats experienced a reduced total number of secondary and antral follicles, and showed more atretic follicles; whereas basal serum FSH levels were scarcely affected [10]. Furthermore, replacement of T4 and gonadotropins in immature rats with hypothyroidism increased the numbers of corpora lutea and follicles, and reinstated luteal angiogenesis [42]. Our results clearly showed that localization of TRa1 on PND 10 and 21 in the rat ovary. TRa1 showed strong staining in oocytes of all follicles and in the granulosa and theca cells of follicles. It is interesting to note that we found TRa1 to be strongly expressed and localized in the granulosa cells and theca cells during follicular development on PND 10 and 21 in control rats, and in both hyper and hypo groups. A few previous studies described the expression TRa1 in the ovary, with some staining in oocytes of primordial, primary, secondary, and antral follicle in humans, with slight staining in granulosa cells of secondary follicles [8, 43]. Furthermore, expression of TRa1 was detected in oocytes and germinal vesicles in women [44, 45]. Our present data suggest modulation of expression patterns of NOS isoforms and enzyme activities in neonatal rat ovaries from PND 10 to 21. The NOS expressed in rat ovarian tissues might also be regulated by TH. Earlier studies showed that granulosa cells are crucial for oocyte growth, nuclear meiotic status, cytoplasmic development, and genomic transcriptional activity [15, 46]. In ovarian tissues, nNOS protein shows staining in the oocyte, and granulosa and theca cells of follicles in neonatal normal rats [18]. In addition, in fetal and neonatal pigs, expressions of nNOS and iNOS were detected in the oocytes of primordial, primary, and secondary follicles, and in granulosa cells and theca cells; iNOS was also expressed in blood

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vessels and corpora lutea, and it was weakly localized in the granulosa cells and theca cells of follicles [20]. Immunohistochemical studies demonstrated the expression of iNOS in the ovarian surface epithelium, oocytes, and theca of multilaminar and antral follicles in pigs. In the bovine, eNOS protein was localized in primordial, primary, and antral follicles and in blood vessels [47]. Our results found that total NOS and iNOS activities on PND 10 and 21 were increased in the hyperthyroid rats and reduced in the hypothyroid rats. Previous studies demonstrated recently that TH regulated NOS gene expressions in rat hypothalamus, and also indicated that in hypothyroidism reduced gene expression while hyperthyroidism increased it [48]. Moreover, NOS activity was higher in heart, vessels and kidney of hyperthyroid rats, and was significantly decreased in the same tissues with hypothyroidism [49]. In rat cerebral cortex during postnatal development, T4 significantly increased NOS activity, which was decreased significantly in hypothyroid rats [50]. In women gastroparesis causes a lack of estrogen, which then reduces tetrahydrobiopterin (BH4) and nNOS activity [51]. In women, gastroparesis in the luteal phase of the menstrual cycle alters sex hormones, causing harmful effects [52]. Hypothyroid patients with polycystic ovaries also had lower androstenedione levels compared with those who had normal-appearing ovaries [53]. TH and NO production might be involved in the aforementioned phenomena due to the presence of TH/NOS signaling pathways during the process of ovarian follicular development. In summary, using a rat ovary model, we demonstrated a role for TH during the neonatal critical period on ovarian follicular development. Our observations are expected to further enrich our understanding of the roles THs might play in the regulation of the basal functions in ovarian follicular development. We surmise that there may be involvement of the NOS signaling pathway throughout ovarian follicular development in hyperthyroid and hypothyroid states. NOS activities in the hyper- and hypothyroid ovary require further elucidation. Acknowledgments We express our gratitude to Dr. Reinhold J. Hutz of the Department of Biological Sciences, University of Wisconsin-Milwaukee in USA for reading the original manuscript and offering valuable suggestions. This work was supported by the National Nature Science Foundation of China (No. 31172206). Conflict of interest peting interests.

The authors declare that they have no com-

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