Influence of maternal hypothyroidism on the postnatal development of rat testes as detected by morphometric and lectin histochemistry analysis
Thyroid disorders - the most widespread endocrine pathologies, which influencing multiple organs, testes among others. Characteristic of the impact of maternal hypothyroidism on postnatal development of male reproductive system including its glycom.
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| Язык | английский |
| Дата добавления | 10.05.2018 |
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By means of morphometric and lectin histochemistry analysis to investigate the influence of maternal hypothyroidism on postnatal morphogenesis of rat progeny testes. thyroid disorder postnatal endocrine
Material and Methods. Experimental hypothyroidism was induced in 15 female Wistar rats by supplementation of their daily food allowance with 5 mg/kg of mercazolil during 14 days prior to fertilization and during whole gestational period. Testes of their progeny on prenatal day 20th, postnatal days 1st, 20lh, 40th and 120h were excised, fixed in Bouin's fluid, embedded in paraffin and subjected to morphometric and lectin histochemistry investigation. The obtained data was compared with the same parameters ofprogeny from control group rats. Results and Discussion. Maternal hypothyroidism in prenatal day 20th rats was associated with decreased size and enhanced count of Sertoli cells within the seminiferous cords; decreased amount of interstitial connective tissue combined with significant elevation of Leydig cells count and reduced lectin binding to all testicular components. On postnatal day 1st it was detected the increased Leydig cells content, though less prominent in comparison with prenatal period. On postnatal day 20th size of seminiferous tubules of hypothyroid rats progeny strongly exceeded control parameters due to the increased spermatogenic cells proliferation rate. On postnatal day 40th seminiferous tubules of experimental group rats contained large conglomerates of degenerating and apoptotic spermatogenic cells. In the adult rats, the decreased size of seminiferous tubules coincided with the increased spermatogenic index and content of Leydig cells. The latter were selectively labeled with PSA, GNA and CCRA, mast cells with WGA, early acrosomes with SBA, cap stage and wedged acrosomes with PNA and SNA lectins. Conclusions. Maternal hypothyroidism has significant impact on postnatal morphogenesis of the rat testes. Differences in lectin binding to tissue samples of control and experimental rat testes were most significant in prenatal animals with certain levelling until adulthood, while morphometric indices were clearly distinct in all investigated progeny groups. Documented selective lectin labeling of Leydig cells with lectins PSA, GNA and CCRA, of mast cells with WGA, as well as different stages of acrosomal maturation with SBA, PNA and SNA can be recommended for further morphometric investigations.
Thyroid gland pathology occupies a significant place in the structure of endocrine pathologies with a trend to increase both in the world as a whole, and in Ukraine in particular [1]. Disorders of the thyroid gland affect about 3% of the world's population and the rate of increase in the number of these patients in the past 10 years remains invariably high. Hypothyroidism affects women roughly 20 times more often than men; it is often combined with pregnancy [2, 3], directly affecting children's health [3, 4, 5].
Infertility is an urgent medical and social problem, since according to the statistical data it affects about 20% of marriages, and in 50% of cases it is caused by male factor [6]. Formerly it was believed that thyroid hormones possess faint if any influence on the male reproductive system, but recent studies revealed the important role of triiodothyronine and thyroxine in testicular development and histophysiology [7]. In particular, it was established that hypothyroidism enhances proliferation and inhibits differentiation of Sertoli and Leydig cells, while hyperthyroidism has the opposite effect inhibiting these cells proliferation and accelerating differentiation, which apparently has a significant impact on the process of testicular maturation and histophysiology of spermatogenesis [8].
Numerous publications demonstrate the important role of carbohydrate determinants lectin receptor sites of tissue glycoconjugates in normal histophysiology, as well as their involvement in the development of different pathologies [9-14]. In previous papers we described morphological parameters and testicular glycans profile of rat progeny that developed under physiological conditions [15, 16]. The purpose of this work using morphometric and lectin histochemistry analysis to investigate the influence of maternal hypothyroidism on postnatal morphogenesis of offspring rat testes.
The investigation was conducted on 25 female Wistar rats 180-200 g of weight, which were divided into two groups: first control (10 animals), and second experimental (15 animals), progeny from which included 40 and 35 rats respectively. The animals were kept under standard vivarium conditions, with maintenance of sanitary norms and diet. Experiments were conducted in agreement with the Bioethics Commission of LNMU (protocol № 2 of February 15, 2016) in accordance with the provisions of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (Strasbourg, 1986), Council of Europe Directives 2010/63/ EU, Law of Ukraine № 3447-IV "On the Protection of Animals from Cruel Behavior".
Exogenous hypothyroidism was induced by daily supplementation of experimental group animals' food allowance with 5 mg/kg of mercazolil (Kharkiv, "Zdorovja") during 14 days prior to fertilization and throughout the whole gestational period. After the second week from the beginning of the experiment, the oestral cycle was controlled by the daily taking of vaginal smears from control and experimental rats. In the stage of oestrus rats were fertilized by intact males. The first day of pregnancy was identified by the presence of sperm in the vaginal smears. Verification of hypothyroid status included determination of T3 and T4 hormone levels in blood serum, carried out in the radioisotope laboratory of Lviv regional clinical hospital, as well as histological examination of the thyroid glands: thyrocytes of hypothyroid rats acquired columnar form; follicles contained trace amount or completely lacked colloid.
Table 1
|
Lectin |
Full name / Source |
Carbohydrate specificity* |
|
|
PSA |
Pisum sativum agglutinin |
DMan |
|
|
GNA |
Galanthus nivalis agglutinin |
DMan |
|
|
PNA |
Peanut agglutinin |
DGal(3(1-3)DGalNAc |
|
|
CNFA |
Clitocybe nebularis fungus agglutinin |
DGalNAcP(1-4)DGlcNAc > DGal |
|
|
CCRA |
Cyprinus carpio roe agglutinin |
DGalNAc > DGal |
|
|
SBA |
Soybean agglutinin |
aPDGalNAc |
|
|
MPFA |
Micena pura fungus agglutinin |
DGlcNAcP(1-2)DMana(1-6) |
|
|
WGA |
Wheat germ agglutinin |
DGlcNAc > NeuNAc |
|
|
SNA |
Sambucus nigra agglutinin |
NeuNAca (2-6)DGal |
|
|
LABA |
Laburnum anagyroides bark agglutinin |
aLFuc |
Testes from the offspring of experimental and control group animals were obtained on the 20th prenatal, 1st, 20th, 40th and 120th days of postnatal development. Histological material was fixed in Bouin's fluid and embedded in paraffin. For general morphology investigation sections 5-7 pm thick were stained with haematoxylin and eosin. Morphometric studies included evaluation of seminiferous cords (SC) and seminiferous tubules (ST) number per one field of observation, determining their area), index of spermatogenesis), Leydig cells covered area in one field of observation [6, 17, 18]. Micromorphology of rat testes on the subsequent stages of postnatal morphogenesis was characterized according to Parker and Picut [19], the stages of spermiogenesis were identified according to Leblond and Clermont scale [20].
Glycoconjugates of testicular structures were examined using 10 lectins of different carbohydrate specificity (Table 1), which were purified and conjugated to horseradish peroxidase by Dr. Pharm. Sci, Professor V. Antonyuk. Visualization of lectin receptor sites was performed by 3,3'-diaminobenzidine (Sigma, USA) in the presence of H2O2 as described earlier [15, 16]. Microscopic investigation and photographic work were performed using a "Granum R6053" photomicroscope equipped with an "Echoo-Imager 502000" camera and a "ToupView 3.7" computer program. Morphometric investigations were performed using ImageJ, statistical analysis using Microsoft Office Excel software.
Fertility of hypothyroid rats was lower then that of control group animals: 10 rats of control group delivered 40 viable offspring, while 15 experimental rats delivered only 35 offspring (4.0 and 2.3 per individual respectively). 6% progeny of mercazolil treated group was delivered dead. Total body weight of hypothyroidism affected animals on prenatal day 20th was significantly higher (34.07±1.65 g in experimental versus 22.73±4.07 g in control group, P<0.005).
The obtained morphometric data are summarized in Table 2 and presented in Fig.1-7. On prenatal day 20th histological specimens of control rat testes contained 11.65±0.92 SC in one field of observation in comparison to 14.07±1.25 SC of experimental group animals. Diameters of SC were similar, namely, 57.55±2.80 pm in control and 56.56±2.37 pm in experimental group rats. The discrepancies in count of SC apparently rely on higher amount of connective tissue interstitium in control rats (Fig. 1A, B). Moreover, cells filling SC of hypothyroid rat offspring were smaller and more numerous compared with control samples (Fig. 1 C, D). Since on this ontogenetic stage content of SC is represented primarily by Sertoli cells [19], this observation apparently relay on these cells increased proliferation rate and delayed maturation stipulated by thyroid hormones deficiency as reported by Wagner et al. [8].
Fig. 1
Reactivity of hypothyroid rats testicular components with most of the lectins used, except for SBA and LABA, was significantly lower compared to control samples. However, in the previous publications [15, 16] we reported selective labeling of fetal Leydig cells with PSA and GNA.
These same lectins, as well as CCRA, SBA, SNA and LABA distinctly demonstrated increased count of Leydig cells in hypothyroid rats compared to control group progeny (91.82±1.46 and 8.28±0.37 Leydig cells per one field of observation, respectively).
On the postnatal day 1st it was documented similar count and size of SC in testes of control and experimental group animals: one field of observation contained 22.64±4.72 SC in control and 20.87±1.44 SC in experimental rat specimens, diameters of SC corresponded to 51.82±1.36 pm in control and 52.25±2.69 pm in experimental group rats (Fig. 2). Alongside with the increased content of Leydig cells in hypothyroid rats (Fig. 3 A, B), we detected also the increased count of mast cells, which were rather selectively labeled with CNFA and WGA(Fig. 3 C, D).
Table 2. Results of morphometric measurements
|
Seminiferous cord / tubule |
* Count of Leydig cells per one field of observation M±m |
||||||
|
* Count of SC/ST per one field of Observation M±m |
Diameter (Pm) M±m |
Cross section area (pm2) M±m |
Index of spermatogenesis M±m |
||||
|
Prenatal day 20th |
control |
11.65±0.92 |
57.55±2.80 |
2599.92±130.82 |
- |
8.28±0.37 |
|
|
experiment |
14.07±1.25 |
56.56±2.37 |
2511.24± 122.18 |
- |
91.82±1.46** |
||
|
Postnatal day 1st |
control |
22.64±4.72 |
51.82±1.36 |
2107.97±63.78 |
- |
10.84 ±0.86 |
|
|
experiment |
20.87±1.44 |
52.25±2.69 |
2134.64±88.72 |
- |
45.22±4.75** |
||
|
Postnatal day 20th |
control |
24.44±3.3 6 |
76.31±2.22 |
4571.22±135.88 |
- |
9.84±1.46 |
|
|
experiment |
17.42± 1.16** |
101.73±2.60** |
8123.96±167.41** |
- |
11.82±0.37 |
||
|
Postnatal day 40th |
control |
7.62±0.57 |
162.77±4.59 |
20797.85±618.26 |
3.45±0.18 |
11.84± 1.07 |
|
|
experiment |
6.41±0.72 |
169.04±5.89 |
22431.00±670.18 |
3.82±0.22 |
18.81± 1.3 5 * |
||
|
Postnatal day 120th |
control |
2.22±0.61 |
267.85±10.48 |
56318.74±2318.60 |
3.18±0.36 |
18.78±1.07 |
|
|
experiment |
2.00±0.46 |
224.67±7.48** |
39624.14±1235.42** |
3.84±0.41 |
24.22±1.46** |
Fig. 2
On the postnatal day 20th diameter of ST in experimental animals significantly exceeded the same parameter in control rats: 76.31±2.22 pm in control versus 101.73±2.60 pm in experimental rats. Lectin histochemistry studies revealed decreased lectin labeling of adluminal spermatocytes in within the ST, this phenomenon coinciding with early acrosomal granules staining, supplemented with the increased count and enhanced lectin reactivity of Leydig cells (Fig.4). Due to the enhanced proliferation rate of experimental rats spermatogenic epithelium, some of their enlarged size ST were lacking lumina, which were well presented in control specimens.
On the postnatal day 40th the diameter of ST was 162.77±4.59 pm in control and 169.04±5.89 pm in experimental group animals (difference
insignificant). ST lumina of the latter were filled with large conglomerates of degenerating and apoptotic cells (Fig. 5). Index of spermatogenesis corresponded to 3.45±0.18 in control versus 3.82±0.22 in experimental group rats. Differences in lectin binding included intensive CCRA reactivity with spermatogonia nuclei, as well as SBA and MPFA labeling of acrosomal caps of early spermatids in experimental, but not in control group animals.
In the adult rat testes there was detected certain reduction of ST diameter in experimental (224.67±7.48 pm) versus control group rats (267.85±10.48 pm) (Fig.6). Spermatogenic indices were as follows: 3.18±0.36 in control and 3.84±0.41 in experimental animals. ST demonstrated marked heterogeneity in lectin binding, apparently due to different stages of spermatogenic cycle presented in different segments of ST. Subsequent stages in acrosomal maturation were documented by lectin staining: SBA intensely labeled acrosomal granules and caps (stage 3-6 of spermiogenesis), while PNA and SNA labeled acrosomal caps (stage 7-8 of spermiogenesis) and wedged acrosomes (stage 1014 of spermiogenesis) [20] (Fig. 7). Majority of used
Fig. 3
Fig. 4
Fig 5
Fig. 6
Lectin histochemistry of rat testes on postnatal day 20th: spermatocytes in the adlumenal compartments of seminiferous tubules of hypothyroid rats (B, D) demonstrate reduced lectin reactivity in comparison to control rat samples (A, C): PSA (A, B) and CCRA (C, D) lectin labels. x400.
Micromorphology of the rat testes on postnatal day 40th. Haematoxylin and eosin staining: control (A, C), hypothyroid rat (B, D). x100 (A, B) and x400 (C, D) lectins demonstrated high affinity to Leydig cells cytoplasmic glycoconjugates (Fig.3, 4, 7). We did not manage to detect significant differences in lectin binding to testicular specimens of adult control and experimental rats.
Fig. 7
Our morphometric findings evidence that maternal hypothyroidism induces in progeny rats enhanced proliferation ratio of spermatogenic epithelium, increased size of seminiferous tubules and elevated index of spermatogenesis, these findings apparently coinciding with sperm cell delayed maturation. Offspring of hypothyroid rats demonstrated increased content of Leydig cells, which were selectively labeled with PSA, GNA and CCRA lectins. There was also detected decreased reactivity of experimental animals testicular structures with most of the lectins used in comparison to control rat samples.
Conclusions.
1. Maternal hypothyroidism has significant impact on postnatal morphogenesis of rat testes, and the obtained data extend the existing knowledge on fine mechanisms of these influences.
2. Differences in lectin binding to tissue samples of control and experimental rat testes were significant in prenatal animals with levelling until adulthood, while morphometric indices were clearly distinct in all investigated progeny groups.
3. Documented selective labeling of Leydig cells with lectins PSA, GNA and CCRA, of mast cells with WGA, of early acrosomes with SBA, of cap stage and of wedged acrosomes with PNA and SNA can be recommended for further morphometric investigations.
References
1. Zelinska N.B. Congenital hypothyroidism. Ukrainian Journal of Pediatric Endocrynology 2015, № 1: 67-68.
2. Shelestova L.P. Subclinical hypothyroidism in juvenile pregnant women. Endocrynology 2004, № 1: 53-59.
3. Pasechko N.V., Naumova L.V., Heriak S.M. Pathology of thyroid gland and pregnancy. Scientific Research Herald 2009, № 4: 54-56.
4. Olijnyk N.M., Heriak S.M. Influence of thyroid hypofunction on the frequency of pregnancy and parturitional complications. Scientific Research Herald 2001, № 4: 4142.
5. Belych N.A. Influence of iodine deficiency on maternal and fetal thyroid hormonogenesis. Clinical Endocrynology and Endocrine Surgery 2012, № 1: 3 9-46.
6. Hrytsuliak B.V., Hrytsuliak V.B., Pastuch M.B., Dolynko N.P. Histoand ultrastructural changes in the testes of rats with chronic alcohol intoxication. World of Medicine and Biology 2014, № 2(44): 114-117.
7. Krajewska-Kulak E, Sengupta P. Thyroid function in male infertility. Front. Endocrinol. 2013, 4: 174.
8. Wagner M.S., Wajner S.M., Maia A.L. The role of thyroid hormone in testicular development and function. J. Endocrinol. 2008, 199(3): 351-365.
9. Lutsyk A.D., Detiuk E.S., Lutsyk M.D. Lectins in histochemistry. Lviv: Vyshcha Shkola, 1989. 144p.
10. Antonyuk V.O. Lectins and their raw resources. Lviv: Kvart, 2005.550 p.
11. Sharon N, Lis H. Lectins, 2nd ed. Dordrecht: Springer, 2007. 441 p.
12. Varki A, Cummings R.D., Esko J.D. et al. Essentials of glycobiology. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2009.
13. Roth J. Lectins for histochemical demonstration of glycans. Histochem. Cell Biol. 2011,136: 117-130.
14. Dan X, Liu W. Development and applications of lectins as biological tools in biomedical research. Med.Res.Rev. 2015, 36: 221-247.
15. Shegedin A.Yu, Yashchenko A.M., Lutsyk A.D. Exposure of glycoconjugates in postnatal morphogenesis of the rat testis as detected by PAS-reaction and lectin histochemistry. World of Medicine and Biology 2017, № 2(60): 174-178.
16. Shegedin A, Yashchenko A, Lutsyk A. Modification of rat testis carbohydrate determinants in postnatal morphogenesis as detected by lectin probes. J Cell Sci Ther 2017, 8: 274.
17. Pastuchova V.A. Morphological investigation of spermatogenesis in adult rats. Herald of Biology and Medicine 2011, V.2(88): 145-146.
18. Dudenkova N.A., Shubina O.S. Changes of morphofunctional status and productivity of seminal glands of the rats subjected to lead acetate. Fundamental Research 2013, №10 (part 6): 1253-1259.
19. Parker G.A., Picut C.A., eds. Atlas of histology of the juvenile rat. Amsterdam: Elsevier-Academic Press, 2016: 227-247.
20. Leblond C.P., Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann NY Acad Sci, 1952, 55: 548-573.
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