Amitabh Krishna

Localization of Gonadotropin-Releasing Hormone (GnRH), Gonadotropin-Inhibitory Hormone (GnIH), Kisspeptin and GnRH Receptor and Their Possible Roles in Testicular Activities From Birth to Senescence in Mice

The changes in distribution and concentration of neuropeptides, gonadotropin-releasing hormone (GnRH), gonadotropin-inhibitory hormone (GnIH), kisspeptin, and gonadotropin-releasing hormone receptor (GnRH-R) were evaluated and compared with reproductive parameters, such as cytochrome P450 side-chain cleavage (P450 SCC) enzyme activity, androgen receptors (AR) in the testis and serum testosterone levels, from birth to senescence in mice. The results showed the localization of these molecules mainly in the interstitial and germ cells as well as showed significant variations in immunostatining from birth to senescence. It was found that increased staining of testicular GnRH-R coincided with increased steroidogenic activity during pubertal and adult stages, whereas decreased staining coincides with decreased steroidogenic activity during senescence. Similar changes in immunostaining were confirmed by Western/slot blot analysis. Thus, these results suggest a putative role of GnRH during testicular pubertal development and senescence. Treatment with a GnRH agonist ([DTrp(6), Pro(9)-NEt] GnRH) to mice from prepubertal to pubertal period showed a significant increase in steroidogenic activity of the mouse testis and provided further support to the role of GnRH in testicular pubertal maturation. The significant decline in GnRH-R during senescence may be due to a significant increase in GnIH synthesis during senescence causing the decrease in GnRH-R expression. It is considered that significant changes in the levels of GnRH-R may be responsible for changes in steroidogenesis that causes either pubertal activation or senescence in testis of mice. Furthermore, changes in the levels of GnRH-R may be modulated by interactions among GnRH, GnIH, and kisspeptin in the testis.

Immunohistochemical localization of GnRH and RFamide-related peptide-3 in the ovaries of mice during the estrous cycle

Gonadotropin releasing hormone (GnRH) has now been suggested as an important intraovarian regulatory factor. Gonadotropin inhibitory hormone (GnIH) a hypothalamic dodecapeptide, acts opposite to GnRH. GnRH, GnIH and their receptors have been demonstrated in the gonads. In order to find out the physiological significance of these neuropeptides in the ovary, we aim to investigate changes in the abundance of GnRH I and GnIH in the ovary of mice during estrous cycle. The present study investigated the changes in GnRH I, GnRH I-receptor and RFRP-3 protein expression in the ovary of mice during estrous cycle by immunohistochemistry and immunoblot analysis. The immunoreactivity of GnRH I and its receptor and RFRP-3 were mainly localized in the granulosa cells of the healthy and antral follicles during proestrus and estrus and in the luteal cells during diestrus 1 and 2 phases. The relative abundance of immunoreactivity of GnRH I, GnRH I-receptor and RFRP-3 undergo significant variation during proestrus and thus may be responsible for selection of follicle for growth and atresia. A significant increase in the concentration of RFRP-3 during late diestrus 2 coincided with the decline in corpus luteum activity and initiation of follicular growth and selection. In general, immunolocalization of GnRH I, GnRH I-receptor and RFRP-3 were found in close vicinity suggesting functional interaction between these peptides. It is thus, hypothesized that interaction between GnRH I-RFRP-3 neuropeptides may be involved in the regulation of follicular development and atresia.

Effects of gonadotropin-inhibitory hormone on folliculogenesis and steroidogenesis of cyclic mice

OBJECTIVE To evaluate the effects of gonadotropin-inhibitory hormone (GnIH) treatment on ovarian activity of mice. DESIGN Animal study. SETTING Reproductive physiology laboratory of university department of zoology. ANIMAL(S) Twelve-week-old female mice of inbred Parkes strain. INTERVENTION(S) Mice treated with different doses of GnIH (control, 100 ng, 500 ng, and 2 μg per day) for 8 days were studied. For in vitro study, the ovaries of proestrus mice were cultured with different doses of GnIH for 24 hours at 37 °C. MAIN OUTCOME MEASURE(S) Folliculogenesis, steroidogenesis, luteogenesis, and apoptosis in the ovaries of control and GnIH-treated mice. RESULT(S) GnIH treatment produced significant changes in body mass, circulating steroid levels, and ovarian activity in the mice. GnIH also caused dose-dependent histologic changes in follicular development and luteinization. The antral follicles showed abnormal changes. The mice treated with increasing dose of GnIH showed significant changes in steroid synthesis owing to inhibitory effects of GnIH on ovarian expression of LH receptor, steroidogenic acute regulatory, and 3β-hydroxysteroid dehydrogenase proteins. CONCLUSION(S) GnIH inhibited follicular development and steroidogenesis in the ovary of mice. This study thus suggests biologic significance of this neuropeptide in regulating ovarian activity.

Relative role of olfactory cues and certain non-olfactory factors in foraging of fruit-eating bats

The proportion of captivated Indian short-nosed fruit bats, Cynopterus sphinx, approaching fruits within a specified duration and the fruit-approaching latency were recorded under various test conditions (involving variations in the olfactory and certain aspects of the physical environment, and the time of food presentation). While alterations in the majority of physical features in the immediate environment of bats failed to affect either of the foraging parameters studied, one or both of them were markedly influenced by the presence of light or source of a novel odour, or a delay of 24 h in the regular feeding time. When paper pieces coated with homogenized fruit pulp were provided instead of the fruit pieces during the regular feeding time, bats responded normally; they, however, did not approach water-wetted papers. Initially the bats did not consume grapes. However, following the supply of grapes coated with homogenate of banana fruits, grapes were approached and consumed in considerable quantities. The results underscore that light has a crucial role in the foraging activity of C. sphinx and they rely extensively on olfactory cues to detect the fruits. In another experiment the bats were daily provided simultaneous access to two adjacent fruit-containing cages; fruits in one of the cages were accessible but those in the other cage were not. Cedar wood oil, source of a strong odour, was placed in either of the cages. Observations revealed that the bats can learn to associate the availability of fruits with the presence of an odour within 3 days.

Seasonal changes in circulating serum concentration and in vitro testicular secretion of testosterone and androstenedione in the male vespertilionid bat (Scotophilus heathi)

Seasonal changes in the testicular activity were studied in the male Greater Yellow bat, Scotophilus heathi, in Varanasi, India (25 degrees N, 83 degrees E). The seasonal pattern of body weight of male bats was also recorded and compared with circulating androstenedione (A) and testosterone (T) concentration. There was a gradual increase in the body weight of the bat from September onward mainly due to accumulation of adipose tissue. Finally, body weight reached a peak level in November. Two periods of peak testicular weight and serum T concentration but only one peak of serum A concentration were observed. The first peak of T coincided closely with the peak A concentration and increased body weight. The first peak of T occurs several months before the time of mating and ovulation. The second peak of T correlated closely with the period of mating. In vitro study showed seasonal variation of the testicular responsiveness to LH stimulation. In vitro study also showed two peaks in the T but only one peak in the A production by testes in response to LH during different phases. Preliminary study showed that IGF-I but not insulin augmented hCG induced T production by testes in vitro. Our observation of an increase in serum A concentration during the period of overfeeding and weight gain and decline in A during the period of weight loss in S. heathi adds strength to the concept that nutritional status can influence hormonal function. This may be the reason for asynchrony between male and female gametic cycle found among vespertilionid bats.

Effects of GnRH agonist treatment on steroidogenesis and folliculogenesis in the ovary of cyclic mice

BackgroundGnRH analogs (both agonist and antagonist) have been extensively used for clinical applications, following the discovery of its direct effects on ovary. With regard to the direct actions of GnRH agonist on ovary, conflicting data are reported. The mechanism through which GnRH agonist affect gonadal functions is still obscure. The aim of present study was thus to investigate the effects of treatment with different doses of GnRH agonist, in vivo and in vitro, on morphological, physiological and functional changes in the ovary of cyclic mice.MethodsTo find out the effect of GnRH agonist on ovarian activity, cyclic mice were treated with different doses for 8 days and its effect on folliculogenesis (morphological changes in follicle, Estrogen receptor, progesterone receptor), steroidogenesis (circulating progesterone level, StAR, LH-receptor, 3β-HSD), luteinization (Morphology of corpus luteum) and apoptosis (caspase-3, PARP) were observed. To find the in vitro effects of GnRH agonist with or without LH on ovary of mice, changes in the expression of LH-receptor, estrogen receptor, progesterone receptor, 3β-HSD in the ovary and progesterone level in the culture media were investigated.ResultsGnRH agonist treatment produced significant changes in ovarian mass, circulating steroids level and ovarian follicular development, steroidogenesis and apoptosis in the mice. GnRH agonist also caused dose dependent histological changes in follicular development and luteinization. The mice treated with different doses of GnRH agonist showed biphasic effects on steroid synthesis due to its effects on ovarian expression of LH-receptor, StAR, and 3β -hydroxysteroid dehydrogenase proteins. The high dose showed stimulatory effect, whereas pharmacological dose showed inhibitory effect on ovarian follicular development and steroidogenesis. The in vitro study generally showed inhibitory effects of GnRH agonist on ovarian activities, which may be reversed by the presence of LH.ConclusionBoth inhibitory and stimulatory effects found in the present study suggest that GnRH agonist is a versatile tool in the therapy of a variety of gynecological and non-gynecological conditions. This study suggests that the outcome of direct effect of GnRH-ag on ovary depends on LH-responsiveness.

Melatonin modulates glucose homeostasis during winter dormancy in a vespertilionid bat, Scotophilus heathi

The role for melatonin in glucose homeostasis and insulin resistance is not very clear and has recently been an active area of investigation. The present study investigated the role of melatonin in seasonal accumulation of adipose tissue in Scotophilus heathi, with particular reference to its role in glucose homeostasis and development of insulin resistance. The circulating melatonin levels correlated positively (p<0.05) with the changes in body mass due to fat accumulation and circulating insulin level, but correlated negatively with the blood glucose level in S. heathi. The bats showed high circulating blood glucose levels and impaired glucose tolerance during the period of fat deposition suggesting insulin resistance condition which improves after winter when most of the fat has been utilized as a metabolic fuel. The high circulating melatonin levels during the period of maximum body fat at the beginning of winter prepare the bats for winter dormancy by modulating the glucose homeostasis through affecting blood glucose levels, muscle and liver glycogen stores, insulin receptor and glucose transporter 4 (GLUT 4) expression. This is also confirmed by in vivo study in which melatonin injection improves the glucose tolerance, increases muscle insulin receptor and GLUT 4 expression, and enhances glucose clearance from the blood. The results of present study further showed that the effect of melatonin injection on the blood glucose levels is determined by the metabolic state of the bats and may protect from decrease in blood glucose level during extreme starvation, however, melatonin when injected during fed state increases glucose clearance from the blood. In summary, the present study suggested that melatonin interferes with the glucose homeostasis through modulating intracellular glucose transport and may protect bats from hypoglycemia during winter dormancy.

Seasonal adiposity, correlative changes in metabolic factors and unique reproductive activity in a vespertilionid bat, Scotophilus heathi.

The aim of this study was to determine the effect of changes in body mass, fat reserves and feeding activity on circulating levels of lipids, glucose, protein and metabolic hormones in a vespertilionid bat, Scotophilus heathi. Furthermore, the relationship between changes in metabolic factors and hormones with the unique reproductive features of female S. heathi was also examined. The results of this study showed annual variation in body mass, fat reserve and feeding activity, which correlated significantly with circulating levels of lipids, protein and metabolic hormones. Increased corticosterone level during September-October in S. heathi promotes increased feeding activity, which in turn induces hyperinsulinemia in S. heathi during November. Hyperinsulinemia together with low body temperature in November facilitates fat accumulation in bat. Coinciding with the period of fat accumulation raises serum leptin level, which has been demonstrated to suppress ovarian activities thus causing delayed ovulation in S. heathi. Circulating levels of lipids were high during winter dormancy, which may provide energy to stored sperms. The study thus suggests that the unique reproductive features of female vespertilionid bat are strongly linked to fat deposition.

Localization of gonadotrophin-releasing hormone I, bradykinin and their receptors in the ovaries of non-mammalian vertebrates.

GnRH I and its receptors have been demonstrated in the ovaries of various vertebrates, but their physiological significance in reproductive cascade is fragmentary. Bradykinin is a potent GnRH stimulator in the hypothalamus. In the present study, the presence of GnRH I and its receptor, and bradykinin and its receptor in the ovaries of non-mammalian vertebrates were investigated to understand their physiological significance. GnRH I immunoreactivity in the ovaries of fish, frog, reptile and bird were mainly found in the oocyte of early growing follicles and granulosa cells and theca cells of previtellogenic follicles. Vitellogenic follicles showed mild GnRH immunoreactivity. GnRH I-receptor and bradykinin were localized in the same cell types of the ovaries of these vertebrates. The presence of GnRH I, GnRH I-receptor and bradykinin in the ovaries of these vertebrates was confirmed by immunoblotting. The presence of GnRH I mRNA was demonstrated in the ovary of vertebrates using RT-PCR. The ovaries of reptiles and birds showed significantly higher intensity of immunoreactivity for GnRH I-receptor as compared with the fish and amphibian. This may have a correlation with the higher yolk content in the ovary of reptile and bird. These results suggest the possibility of GnRH I and bradykinin as important regulators of follicular development and vitellogenesis in the vertebrate ovary.

Changes in serum leptin, insulin, androstenedione and luteinizing hormone during ovarian cycle in the bat, Taphozous longimanus

Detailed reproductive pattern and associated endocrine characteristics have been documented in only a few species of order Chiroptera. The aim of the present study was to examine the changes in body weight, serum insulin, leptin, androstenedione and luteinizing hormone (LH) concentrations during annual ovarian cycle in the sheath-tailed bat, Taphozous longimanus. Bats were sampled over three years. Leptin, a satiety hormone produced primarily by adipose tissue, provides information to feeding center of the brain about nutritional status, fat mass, appetite and energy expenditure. The circulating concentration of leptin begins to increase from October and attains a peak in December. The peak serum leptin concentration coincides with body weight in November before winter dormancy in December. The serum leptin levels dissociate from body weight during December. The other peaks of serum leptin levels coincide with late stages of the two successive pregnancies. The serum insulin concentration begins to increase from September and attains a peak during December. The insulin concentration remains low from January to August. The circulating androstenedione concentration begins to increase in October, reaching a peak in December. This increase in androstenedione concentration correlated with the period of heavy accumulation of abdominal fat and increase in body weight. There was a sharp decline in androstenedione concentration and body weight in January. The serum LH shows peaks, in November, coinciding with the peaked body weight, the other peaks in January and May, coinciding with ovulation for the two successive pregnancies. The high leptin and insulin levels might be responsible for the maintenance of reproductive response and gonadal function during adverse environmental condition in the winter, while high androstenedione, and associated body weight along with LH might be responsible for maintaining basal gonadal function. We conclude that high leptin, androstenedione and insulin serve, as signal for the reproductive functions in that sufficient body fat are available to meet the caloric demands and maintain normal function during adverse winter conditions.