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1 Seasonal Variation in Thyroid Gland of the Female Bat, Taphozous 2 Kachhensis (Dobson) During Pregnancy 3 Pankaj Chavhan1, Amir Dhamani2 4 1 Department of Zoology, N.H.College, Bramhapuri 441206 5 2 Department of Zoology, S. S. S. Sci. College, Ashti 442707 6 Pankaj Chavhan: [email protected], Amir Dhamani: [email protected] 7 Abstract The aim of present study is to compare the changes in thyroid gland during the 8 reproductive cycle of the female bat Taphozous kachhensis. The thyroid gland of the bat is 9 studied morphometrically and histologically, it showed marked seasonal variation in weight, 10 quantity of colloid, epithelial cell height and plasma concentration of thyroid hormones. The 11 thyroid gland is a symmetrical, bilobed, and located on the lateral side of the trachea, between 12 the first and the third tracheal rings, connected by an isthmus. The mean weight of the noted 13 lobes was (2.240 ± 0.121) mg, (2.88 ± 0.05) mg and (2.704 ± 0.067) mg, during early pregnancy, 14 mid pregnancy and late pregnancy respectively. The thyroid hormone shows marked seasonal 15 variations. During early pregnancy there is rise in the level of TSH (1.7 ng/mL) but the slight 16 decrease in the level of T3 (54 ng/mL) and T4 (2.9 ng/mL) was noticed. The level of TSH and 17 T4 was decreases during mid pregnancy but the increase in the level of T3 was noticed and it is 18 highest as compare to other stages and it is found to be 1.4 ng/mL, 1.5 ng/mL, 69 ng/mL 19 respectively. During late pregnancy, TSH and T3 concentration decreases but the increase in the 20 level of T4 was observed and it is found to be 0.9 ng/mL, 60 ng/mL, 2.9 ng/mL respectively. The 21 diameter of colloid in small, medium and large follicle during early pregnancy and late 22 pregnancy were 28 µm, 56 µm, 96 µm and 36 µm, 60 µm, 72 µm respectively. The epithelial cell 23 heights of small, medium and large size follicles during early pregnancy and late pregnancy were 24 3.6 µm, 4 µm, 8 µm and 4 µm, 8 µm, 8 µm respectively. 25 Keywords Gross anatomy; Histology; Thyroid gland; Bat; T3; T4 26 Introduction 27 The thyroid is an endocrine gland which secretes thyroglobulin, triiodothyronine and thyroxin 28 hormones (Banks, 1993). Thyroxin plays an important role in metabolism of the animal body. 29 Thyroid gland consists of follicles lined by follicular cells. The interfollicular space has been 30 reported to be filled with connective tissue (Leeson and Leeson, 1970). The most outstanding 31 feature of the vertebrate thyroid gland is its ability to concentrate large amount of iodine for the 32 synthesis of thyroxin (Turner, 1966). In addition, thyroid hormones appear to play a key role in 33 the expression of events that underlie seasonal reproductive cycles. The thyroid gland has been 34 studied histologically in many seasonally breeding microchiropteran bats (Kwiecinski et al., 35 1991; Damassa et al., 1995). In general, these morphological studies indicate that the thyroid 36 increases activity late in the winter or early spring, is active throughout the summer, and 37 regresses by autumn. Such an activity cycle is commonly found in most seasonally breeding 38 mammals. 39 A number of hormones secreted by various endocrine glands are directly or indirectly involved 40 in normal functioning of the reproductive processes in various species and the thyroid hormone 41 appear to be one of those that are intricately linked with reproduction. Thyroid hormones may 42 influence reproductive activity either by direct action on the gonads or by effects mediated at the 43 level of the hypothalamo-pituitary axis. 44 Thyroid hormones are described as unique because they exert effects within almost every tissue 45 of the body throughout the life of an individual. In humans, thyroid hormone deficiencies have 46 resulted in a number of abnormalities with respect to growth, development, behavior, 47 metabolism and reproduction. The thyroid gland is an important modulator for reproductive 48 functions. The metabolic hormone, thyroxin, T4 is the physiological regulator of energy balance 49 as well as in maintaining normal reproductive function in mammal (Schwartz et al., 1992; Shi 50 and Berrel,1992). They reported that the T3 is essential for mammalian reproduction in cattle, 51 the deficiency of which result into female infertility. Further it has been reported that a reduction 52 in the secretion and plasma levels of gonadotrophins was associated with hypothyroidism 53 (Hagino, 1971; LaRochelle and Freeman, 1974; Buchanan et al., 1977). Likewise, Dunn et al. 54 (1976) have shown that the daily rhythmic release of luteinizing hormone (LH) and prolactin in 55 rats was altered by thyroidectomy. 56 Thyroid hormones have a significant mode of action on seasonal reproduction. In 1994, work by 57 researchers in Michigan implicated thyroid hormones as having a role in decreasing LH secretion 58 at the end of the breeding season, resulting in the normal cessation of follicular activity and 59 ovulation. Along with follicle stimulating hormone (FSH), luteinizing hormone (LH) and 60 indirectly, gonadotropin-releasing hormone (GnRH) are hormones responsible for follicle growth 61 and ovulation. 62 1 Results 63 1.1 Gross Observations 64 Gross studies of thyroid gland of female bat Taphozous kachhensis revealed that the gland was 65 located near the first ring and third ring of trachea and consisted of two lobes. An isthmus 66 connected these lobes to each other. The colour of the gland was reddish brown. 67 1.2 Seasonal Changes in Body, Ovary, and Thyroid Weights 68 The weights (mean ± SEM) of the body, ovary, and thyroid during reproductive phases are 69 shown in Table 1 and Graph 1. There was not much difference between the highest and lowest 70 mean thyroid weight of adult female bats caught during different phases of pregnancy. The 71 thyroid weight showed two peaks coinciding with the peaks of ovary weight during early 72 pregnancy and mid pregnancy. Both thyroid and ovary began to increase in weight from early 73 pregnancy (January to February) and attained a peak during mid pregnancy (Late February to 74 Middle April). Thyroid weights declined during late pregnancy similarly to ovary weight (Graph 75 1). 76 Table 1 The mean weights of the body, ovary, and thyroid during different phases of pregnancy Early pregnancy (mg) Mid pregnancy (mg) Late pregnancy (mg) Thyroid 2.24 ± 0.120 2.88 ± 0.051 2.70 ± 0.067 Ovary 2.38 ± 0.086 2.49 ± 0.075 1.49 ± 0.064 Body 50.00 45.00 39.00 77 78 79 Graph 1 The mean weights of the body, ovary, and thyroid during different phases of pregnancy 80 1.3 Histology 81 The thyroid gland of the female bat is studied histologically; it showed marked seasonal variation 82 in quantity of colloid, epithelial cell height and plasma concentration of thyroid hormones. 83 Diameter of colloid in large, medium and small size follicle of thyroid gland shows variations, 84 with higher value during early pregnancy and lower value during mid pregnancy in large follicle, 85 while in medium size follicle higher value of colloid during late pregnancy and lower during mid 86 pregnancy. Small size follicle shows higher value of colloid during late pregnancy and lower 87 during early pregnancy (Table 2). Thyroid follicular epithelial cells height also showed marked 88 variations during various period of pregnancy. The large follicles were lined by low cuboidal 89 epithelium having flattened nuclei and 3.6 µm, 4 µm and 4 µm in diameter during early, mid and 90 late pregnancy respectively and were assumed to be inactive. The medium size follicles were 91 lined by cuboidal epithelium having darkly stained nuclei and 4 µm, 8 µm and 8 µm in diameter 92 during early, mid and late pregnancy respectively. The small size follicles were lined by high 93 cuboidal epithelium with rounded nuclei having diameter 12 µm, 12 µm and 8 µm during early, 94 mid and late pregnancy respectively(Figure1-Figure 9,Table 2, Geaph 2). 95 Table 2 The mean diameter of colloid and epithelial cell height of thyroid during different phases of pregnancy Early pregnancy Middle pregnancy Late pregnancy Large Medium Small Large Medium Small Large Medium Small follicle follicle follicle follicle follicle follicle follicle follicle follicle 96.0 56 28 64 40 28 72 60 36 3.6 4 12 4 8 12 4 8 8 Diameter of colloid Epithelial cell height 96 97 98 Figure 1 Figure 2 Figure 3 99 100 101 Figure 4 Figure 5 Figure 6 102 103 104 Figure 7 Figure 8 Figure 9 105 Figure 1 Large follicle during early pregnancy showing low cuboidal epithelium. 106 Figure 2 Medium size follicle during early pregnancy showing cuboidal epithelium. 107 Figure 3 Small size follicle during early pregnancy showing high cuboidal epithelium. 108 Figure 4 Large follicle during mid pregnancy showing low cuboidal epithelium. 109 Figure 5 Medium size follicle during mid pregnancy showing cuboidal epithelium. 110 Figure 6 Small size follicle during mid pregnancy showing high cuboidal epithelium. 111 Figure 7 Large follicle during late pregnancy showing low cuboidal epithelium. 112 Figure 8 Medium size follicle during late pregnancy showing cuboidal epithelium. 113 Figure 9 Small size follicle during late pregnancy showing high cuboidal epithelium. 114 115 116 117 118 119 120 121 122 123 Graph 2 The mean diameter of colloid and epithelial cell height of thyroid during different phases of pregnancy 124 1.4 Plasma Concentration of Thyroid Hormone 125 The thyroid hormone examined during different stages of reproductive cycle of bat Taphozous 126 kachhensis are described below. 127 The thyroid hormone shows marked seasonal variations. During early pregnancy there is rise in 128 the level of TSH (1.7 ng/mL) but the slight decrease in the level of T3 (54 ng/mL) and T4 (2.9 129 ng/mL) was noticed. The level of TSH and T4 was decreases during mid pregnancy but the 130 increase in the level of T3 was noticed and it is highest as compare to other stages and it is found 131 to be 1.4 ng/mL, 1.5 ng/mL, 69 ng/mL respectively. During late pregnancy, TSH and T3 132 concentration decreases but the increase in the level of T4 was observed and it is found to be 0.9 133 ng/mL, 60 ng/mL, 2.9 ng/mL respectively (Table 3,Graph 3). 134 135 Table 3 Hormonal concentration of Thyroid hormone and TSH during various phases pregnancy Concentration of hormone (ng/mL) Reproductive period TSH ±SEM T3 ±SEM T4 ±SEM Early pregnancy 1.7 ±0.13 54 ±1.80 2.9 ±0.14 Mid-pregnancy 1.4 ±0.12 69 ±1.91 1.5 ±0.05 Late-pregnancy 0.9 ±0.09 60 ±1.28 2.9 ±0.11 136 137 138 139 140 141 142 143 144 145 146 Graph 3 Hormonal concentration of Thyroid hormone and TSH during various phases pregnancy 147 2 Discussion 148 Thyroid is an endocrine gland that secretes hormones including thyroglobulin, triiodothyronine 149 and thyroxin. Thyroxin hormone secreted by this gland plays an important role in metabolism of 150 the body (Turner, 1966). It is a metabolically important gland, which is a suggested to be 151 essential for the normal maintenance of reproductive function, impairment of thyroid activity 152 may be inhibitory to reproduction (Peebles et al., 1984; Jannini et al., 1995). 153 Pregnancy alters thyroid status in rodents. In the rat, pregnancy results in decreased total T4 and 154 T3 concentrations and enlarged thyroid gland volume. However, unlike the case of humans, 155 iodine up take is decreased in pregnant rats, and urinary iodide excretion remains unaltered the 156 last days of gestation (Calvo et al., 1999, Feldman, 1958. Versloot et al., 1997). 157 In Taphozous kachhensis, the location of thyroid gland in the body is similar to other large 158 animals like cattle and buffaloes (Getty et al., 1986) and Camel (Kausar et al., 2006) i.e., with 159 the first ring of trachea and consisted of two lobes on both side and an isthmus connecting these 160 lobes. In Taphozous kachhensis the gland appeared reddish brown in colour which is in 161 concordance with the findings of Schwartz and Dioli (1992). The present study also showed a 162 close relationship between changes in the weight and morphological features of the thyroid and 163 the ovarian cycle of Taphozous kachhensis. 164 Histological studies revealed similar results as reported in camel (Abdel-Magied et al, 2000; 165 Atoji et al., 1999) i.e. the gland consisted of follicles of variable sizes with small size follicle 166 lined by high cuboidal epithelium to columnar epithelial cells, while the larger ones were lined 167 by low cuboidal epithelial cells. Medium size follicles were lined by cuboidal epithelium. 168 Similar observation were reported in Taphozous longimanus (Nerkar, 2007), Megaderma lyra 169 lyra (Sonwane, 2010). 170 In present study the thyroid hormone show marked seasonal variation. The concentrations of 171 TSH, T3, and T4 show significant seasonal changes. The hormonal level of TSH, T3, T4 changes 172 during early pregnancy and observed1.7 ng/mL, 54ng/mL and 2.9ng/mL respectively. During 173 mid pregnancy the T3 level is found to be 69ng/mL and 60ng/mlL during late pregnancy. T4 174 level is highest during early and late pregnancy (2.9 ng/mL) and lowest during mid-pregnancy 175 (1.5ng/mL). The T3 level is higher than T4 level during reproductive cycle. The serum T3 and 176 T4 concentration showed significant variation with changes in reproductive cycle of bat, 177 Taphozous longimanus (Singh et al., 2002). 178 The thyroid hormones are important for regulation of nutrient assimilation, metabolism, 179 calorigenesis (Todini et al, 2007), reproduction (Blaszczyk et al, 2004). The report of thyroid 180 hormone on pregnancy was reported in Macrotus californicus (Burn et al., 1972). Thyroid 181 hormone plays a important role in the central regulation of body temperature, stimulating the 182 thermogenesis and regulating cellular metabolism (Seitz et al., 1985). The metabolism hormone 183 Thyroxin (T4) has been implicated in the physiological regulation of energy balance as well as 184 maintaining normal reproductive function in mammals (Boswell et al., 1994). 185 In ewes, plasma T4 levels is lower during the luteal phase; T3 concentrations were higher during 186 the luteal phase, This observation supports the present study. 187 During pregnancy, thyroid activity and circulating hormone levels are reported to increase in all 188 the investigated mammalian species. Several mechanisms have been claimed to explain these 189 observations: increased binding protein concentrations in plasma, secretion of thyrotropic factors 190 by the placenta, enhanced responsiveness of pituitary TSH secretion to hypothalamic TRH and 191 changes in maternal TH catabolism (De Leo et al., 1998; Glinoer, 2001).Towards the end of 192 pregnancy, the goat foetus(es) should play a competitive role (higher thyroid activity, iodine 193 affinity and uptake than maternal ones), so that a decrease in maternal plasma T4 concentrations 194 has been observed (McDonald et al., 1988). 195 Plasma T4 concentration was highest during early pregnancy and decreased gradually, reaching 196 lowest values during late pregnancy and post partum (Assane and Sere, 1990; Okab et al., 1993; 197 Yildiz et al., 2005). Like in goats, maternal T3 and T4 in twin pregnancy were lower compared 198 with single-bearing sheep (Yildiz et al., 2005), especially at the end of pregnancy (Assane and 199 Sere, 1990). 200 3 Material and Methods 201 All bats were trapped alive from Ambai-Nimbai adjacent to Kampa-Tempa . Body weight of 202 each bat was recorded. Based on the reproductive cycle of Tapozous kachhensis, females were 203 classified into the following three stages: 204 Early pregnancy (January to February): Ovary shows well developed introvert corpus luteum 205 occupied ¾ part of ovary. 206 Mid pregnancy (Late February to Mid April): Ovary shows well developed introvert corpus 207 luteum occupied entire ovary except small peripheral region. 208 Late pregnancy (Late April to Late May): Ovary shows regress corpus luteum with shrunken 209 luteal cells. 210 1.1 Collection of Serum and Tissues Histology 211 The female bats were sacrificed as soon as they arrived in the laboratory. Their blood serum was 212 collected and assayed in laboratory. Ovary and thyroid were excised out from the body cavity 213 and excess fat and connective tissue attached were separated out. All the tissues were fixed in 214 Alcoholic Bouin’s fluid for 24 h, followed by preservation in 70% ethyl alcohol. Each tissue was 215 weighed separately after placement in 70% alcohol. The tissues were dehydrated in ethanol, 216 cleared in xylol, embedded in paraffin wax, serial sections at 6 μm were cut and stained with 217 haematoxylin and eosin. 218 1.2 Morphometry 219 Thyroid follicular epithelial height and diameter of colloid was measured using ocular 220 micrometer. Measurements were taken from at least 5 different randomly selected thyroid 221 follicles. 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