Induction of ovulation by clomiphene citrate in the Indian vespertilionid bat, Scotophilus heathi

The ovulation induction property of clomiphene citrate (CC) and human chorionic gonadotropin (hCG) was studied in Scotophilius heathi, an Indian tropical vespertilionid bat, during the period of delayed ovulation between December to early January. The results of the study showed that 10 microg of CC alone was ineffective to induce ovulation, whereas 100 microg CC and 10 IU hCG alone induced ovulation. A significant (P < 0.01) increase in the ovulation rate was observed when 10 microg CC followed by 10 IU hCG, compared to 10 IU hCG and 100 microg CC alone groups. Finally, CC at a 100 microg dose, followed by 10 IU hCG, produced superovulation (14.00 +/- 0. 70), which is significantly different in comparison to all other groups. This is the first report of ovulation induced by CC in the Indian tropical bat as well as in any animal model that exhibits temporary anovulation similar to polycystic ovary syndrome (PCOD) during the normal physiology of reproduction.     

Relationship between corticosterone and body weight,androstenedione and insulin during the period of delayed ovulation in a vespertilionid bat,Scotophilus heathi

The aim of the present study was to evaluate the relationship between corticosterone, body weight, insulin and androstenedione in order to understand the role of adrenal in contributing hyperandrogenism during delayed ovulation in S. heathi. The circulating corticosterone concentration in female S. heathi showed significant seasonal variation. The peak corticosterone concentration observed during August-September coincides with increased feeding activities in S. heathi. The present study noted a seasonal variation in relationship of corticosterone with insulin and androstenedione in S. heathi. An inverse relationship of corticosterone with insulin and androstenedione was found during August to December, but not during January to May. A seasonal variation in the effect of adrenocorticotropic hormone (ACTH) on adrenal corticosterone production in vitro was observed during reproductive cycle. Corticosterone production in vitro by adrenal declined significantly as compared to the control during quiescence in September. The finding suggests that adrenal attained the peak responsiveness to ACTH during September. ACTH significantly enhanced the androstenedione production by the adrenal in vitro during December, when the circulating androstenedione was also high in S. heathi. This suggests that the adrenal may also contribute to hyperandrogenism during the period of delayed ovulation in S. heathi. Further studies are required to reveal the unique pattern of seasonal relationship between corticosterone, insulin and androstenedione in S. heathi.     

Seasonal adiposity and delayed ovulation in a vespertilionid bat,Scotophilus heathi: role of tumor necrosis factor-alpha

The aim of this article was to evaluate the physiological significance of tumor necrosis factor-alpha (TNF-alpha) in seasonal accumulation of adipose tissue, hyperinsulinemia, and anovulation in Scotophilus heathi. The result showed seasonal variations in the circulating TNF-alpha level. A higher level of circulating TNF-alpha was observed during quiescence and recrudescence, whereas a lower level of TNF-alpha was observed during winter dormancy and the preovulatory period. An increased circulating TNF-alpha level coincided closely with accumulation of adipose tissue and hyperinsulinemia. Immunocytochemical localization of TNF-alpha in the ovary showed immunoreactivity mainly in the oocytes and theca-interstitial cells. The oocytes of small and medium-sized follicles showed strong TNF-alpha immunostaining, whereas weak immunoreactivity was observed in the large antral follicles. The atretic follicles showed mild TNF-alpha immunostaining. TNF-alpha immunoreactivity in the ovary was slightly higher during the quiescence and preovulatory periods compared with the periods of recrudescence and winter dormancy. TNF-alpha alone significantly increased androstenedione and estradiol production by the ovary in vitro but did not augment the luteinizing hormone (LH)-induced androstenedione production. However, TNF-alpha did augment LH-induced estradiol production. The results of this study suggest the involvement of TNF-alpha in the interaction among adipose tissue accumulation, insulin resistance, and ovarian activity in S. heathi.     

Hormonal induction of ovulation stimulates atresia of antral follicles in a vespertilionid bat, Scotophilus heathi

Scotophilus heathi is a seasonally monoestrous subtropical vespertilionid bat found at Varanasi, India. Although the antral follicles remain present in the ovaries of S. heathi from November till March, ovulation is delayed in this species until early March. In order to understand the mechanism of ovulation suppression during this period of delayed ovulation, the effects of human chorionic gonadotropin (hCG), pregnant mare's serum gonadotropin (PMSG), follicle stimulating hormone (FSH) and gonadotropin releasing hormone agonist (GnRH agonist) on ovarian morphology and steroid concentration were investigated. Hormonal treatments were given as a single i.p. dose 24 h after capture. The bats were sacrificed 48 h after the injection. Treatment with hCG, PMSG, FSH and GnRH agonist failed to induce ovulation in S. heathi, although these hormones produced a high degree of ovarian stimulation. The administration of hCG and PMSG induced ovarian enlargement, intense hyperemia, marked changes in the interstitial cells (ICs), development of several antral follicles and a varying degree of abnormalities in the oocytes of most of the antral follicles. In the bats treated with hCG, PMSG and GnRH agonist, androstenedione concentration increased significantly to extraordinarily high levels, whereas estradiol concentration decreased. Administration of FSH caused regression of ICs and pyknosis of granulosa cells in the majority of antral follicles. FSH did not enhance androstenedione concentration. The results of the present study suggest that the failure of hormonal treatments to induce ovulation during the period of delayed ovulation might be due to a seasonal desensitization of ovarian follicles in S. heathi. The hormonal treatment instead stimulated the ICs to produce a high level of androstenedione resulting in atretic changes of the antral follicles.     

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.     

Histochemical localization of enzymes and lipids in the ovary of a vespertilionid bat, Scotophilus heathi, during the reproductive cycle.

The present study describes seasonal changes in delta5 3beta hydroxysteroid dehydrogenase (3beta-HSD), glusose-6 phosphates dehydrogenase (G-6-PD), and lipids in the ovary of a vespertilionid bat, Scotophilus heathi. Total lipids and 3beta-HSD activity are restricted to thecal and interstitial cells of the ovary. The total lipids, 3beta-HSD, and G-6-PD significantly increase during recrudescence, and remain high during winter dormancy and breeding as compared to the other reproductive phases. High incidence of lipids and enzyme activity in interstitial cells during the breeding period and at the time of ovulation clearly suggests that these cells are actively involved in steroidogenesis. A decline in enzymes and lipid activity during winter dormancy, which correlates with the declining levels of steroidogenesis, might be the factors responsible for prolonged survival of the Graafian follicle in the ovary of S. heathi.     

Ovulation induction by antiestrogens in an Indian tropical vespertillionid bat, Scotophilus heathi

The ovulation induction property of ICI 182,780 a pure antiestrogen and enclomiphene citrate (ENC) was carried out in Scotophilus heathi, an Indian tropical vespertillionid bat, during December to February i.e., preovulatory period. This bat ovulates two ova naturally and shows ovulatory asynchrony. The study showed that 100 ìg of ENC followed by 10 IU hCG resulted in significantly lower number of ovulation. Whereas, the pure antiestrogen ICI 182,780 at a dose of 100 ìg followed by 10 IU hCG resulted in ovulation induction (4.2 +/- 0.4), which is significantly different in comparison to other groups. This is possibly the first report of ovulation induction using this pure antiestrogen i.e., ICI 182,780 in any bat as well as in any animal model that exhibits temporary anovulation similar to polycystic ovary disease (PCOD). This antiestrogen may be useful to induce ovulation in PCOD patients.     

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. J. Exp. Zool. 309A:94-110, 2008. (c) 2008 Wiley-Liss, Inc.     

Mechanism of delayed ovulation in a vespertilionid bat, scotophilus heathi: role of gonadotropin, insulin, and insulin-like growth factor-1

The aim of this study was to evaluate the effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), insulin, and insulin-like growth factor-1 (IGF-1) on ovarian androstenedione synthesis to understand the mechanism responsible for delayed ovulation in Scotophilus heathi. We found that LH stimulated a dose-dependent increase in androstenedione synthesis by the ovary in vitro. This study also showed a clear seasonal variation in the ability of the ovary to produce androstenedione in vitro in response to LH and FSH stimulation. In response to LH and FSH, maximum quantities of androstenedione were produced during recrudescence in November. The same doses of gonadotropins during the preovulatory period in February stimulated comparatively low androstenedione secretion by the ovary. On the basis of these data, we suggest that in S. heathi, ovarian responsiveness to LH and FSH peaks during recrudescence. This study also showed a seasonal variation in the effects of insulin and IGF-1 on ovarian androstenedione production in vitro. Peak ovarian responsiveness to insulin and IGF-1 was observed during quiescence in September. It is hypothesized that increased insulin/IGF-1 sensitivity during September may be responsible for increased responsiveness to LH. Increased LH release, if coincident with the period of enhanced ovarian responsiveness to LH, may result in the excessive androstenedione production responsible for delayed ovulation in S. heathi.     

Immunolocalization of cytochrome P450 side chain cleavage, 17-alpha-hydroxylase and aromatase in the ovary of vespertilionid bat (Scotophilus heathi) during different phases of ovulatory delay.

Immunocytochemical localization of steroidogenic enzymes, cytochrome P450 side chain cleavage, 17-alpha-hydroxylase and aromatase, was performed in the ovaries of Scotophilus heathi during reproductive cycle, with reference to the period of delayed ovulation. Moderate immunoreactivity of side chain cleavage enzyme and 17-alpha-hydroxylase was observed mainly in thecal cells and interstitial cells of the ovarian stroma during quiescence. Thecal cells positive for 17-alpha-hydroxylase were found even around the primary follicles. The peak immunoreactivity for all the three enzymes was observed during recrudescence. It coincided with high circulating steroid levels during this period. In the stroma, immunoreactivity for side chain cleavage and 17-alpha-hydroxylase was so extensive that it almost occupied the entire interfollicular area of the ovary. Aromatase immunoreactivity declined, but side chain cleavage enzyme and 17-alpha-hydroxylase remained extensive during the period of delayed ovulation. This suggests a high androgen and low estrogen synthesis during the period of delayed ovulation. There was a marked decline in 17-alpha-hydroxylase and an increase in aromatase immunoreactivity during the preovulatory period, suggesting a decrease in androgen and increase in estrogen synthesis. The results suggest thecal cells and interstitial cells of the stroma as the major site of steroidogenesis in the ovary of S. heathi. Over production of androgen is attributed to the extensive development of 17-alpha-hydroxylase positive interstitial cells in the ovarian stroma, and this may be responsible for delayed ovulation in Scotophilus heathi.     

Adiposity and Androstenedione Production in Relation to Delayed Ovulation in the Indian bat,Scotophilus heathi

Observations on body weight, circulating androstenedione concentrations and morphology of ovarian stroma were made in Scotophilus heathi during the period of delayed ovulation to make a comparison with polycystic ovarian syndrome in women. Body weight of bats increased from a level of 31.00 ± 0.30 g in August and reached a peak of 45.00 ± 0.46 g in November. This increase in body weight was due to accumulation of adipose tissue. The body weight declined gradually from December onwards and finally reached a basal level in March. The circulating androstenedione concentration showed a gradual increase from 36.80 ± 15.54 ng/ml in August and reached a peak level of 220.50 ± 50.10 ng/ml in November. Androstenedione concentration reached the lowest level in the March, just before ovulation. Morphological study showed extensive distribution of luteinized stromal cells or interstitial cells (ICs). Morphometric study showed that during the period of ovulatory delay, more than 75% area of the ovary was occupied by the ICs. Hyperandrogenism, anovulation, obesity (fat deposition) and stromal hyperthecosis present during delayed ovulation in S. heathi may serve as an experimental model for some aspects of the polycystic ovarian condition in women.     

Seasonal adiposity and androstenedione production as a possible mechanism for asynchronous reproductive activity between males and females of vespertilionid bat, Scotophilus heathi

Relationship between changes in the body weight and circulating androstenedione concentration was examined in order to find out their involvement in asynchronous gametic cycle of male and female S. heathi. Body weight of both male and female bats increased from September due to accumulation of white adipose tissue and reached to a peak in November. The body weight declined gradually due to consumption of adipose tissue during December and January reaching basal level in February. A significant correlation between body weight and serum androstenedione concentration was observed. Correlated with maximum body weight, peak androstenedione level was found during November in both male and female bats. Peak androstenedione level during November may be responsible for stimulating spermatogenesis in the male, but may suppress follicular growth and ovulation in the female, thus resulting in asynchronous gametic cycle of male and female S. heathi.     

Pattern of ovarian protein synthesis and secretion during the reproductive cycle of Scotophilus heathi: synthesis of an albumin-like protein.

Ovarian proteins synthesized de novo and secreted in vitro were investigated during different stages of the reproductive cycle of Scotophilus heathi. We found increased ovarian protein synthesis and secretion during the recrudescence and the preovulatory periods, coinciding with two peaks of follicular development and steroidogenesis. The ovaries synthesized protein at a low rate during quiescence and the late phase of delayed ovulation. In vitro analysis using 35 S-methionine incorporation showed increased synthesis of 66 kDa protein during delayed ovulation, but secretion of this protein declined markedly during the preovulatory period. N-terminal sequencing of the 66 kDa protein showed that it shares 70% homology with human serum albumin. Immunocytochemistry indicated that this protein is found primarily in the granulosa cells of the follicles. Whether the increase in albumin production by the ovaries during delayed ovulation is associated with a hyperinsulinemic and hyperandrogenic condition requires further investigation.     

Pattern of ovarian protein synthesis and secretion during the reproductive cycle of Scotophilus heathi: synthesis of an albumin-like protein

Ovarian proteins synthesized de novo and secreted in vitro were investigated during different stages of the reproductive cycle of Scotophilus heathi. We found increased ovarian protein synthesis and secretion during the recrudescence and the preovulatory periods, coinciding with two peaks of follicular development and steroidogenesis. The ovaries synthesized protein at a low rate during quiescence and the late phase of delayed ovulation. In vitro analysis using ³⁵S-methionine incorporation showed increased synthesis of 66 kDa protein during delayed ovulation, but secretion of this protein declined markedly during the preovulatory period. N-terminal sequencing of the 66 kDa protein showed that it shares 70% homology with human serum albumin. Immunocytochemistry indicated that this protein is found primarily in the granulosa cells of the follicles. Whether the increase in albumin production by the ovaries during delayed ovulation is associated with a hyperinsulinemic and hyperandrogenic condition requires further investigation.     

Pattern of ovarian protein synthesis and secretion during the reproductive cycle of Scotophilus heathi: synthesis of an albumin-like protein

Ovarian proteins synthesized de novo and secreted in vitro were investigated during different stages of the reproductive cycle of Scotophilus heathi. We found increased ovarian protein synthesis and secretion during the recrudescence and the preovulatory periods, coinciding with two peaks of follicular development and steroidogenesis. The ovaries synthesized protein at a low rate during quiescence and the late phase of delayed ovulation. In vitro analysis using ³⁵S-methionine incorporation showed increased synthesis of 66 kDa protein during delayed ovulation, but secretion of this protein declined markedly during the preovulatory period. N-terminal sequencing of the 66 kDa protein showed that it shares 70% homology with human serum albumin. Immunocytochemistry indicated that this protein is found primarily in the granulosa cells of the follicles. Whether the increase in albumin production by the ovaries during delayed ovulation is associated with a hyperinsulinemic and hyperandrogenic condition requires further investigation.     

Effects of reproductive condition,roost microclimate,and weather patterns on summer torpor use by a vespertilionid bat

A growing number of mammal species are recognized as heterothermic, capable of maintaining a high‐core body temperature or entering a state of metabolic suppression known as torpor. Small mammals can achieve large energetic savings when torpid, but they are also subject to ecological costs. Studying torpor use in an ecological and physiological context can help elucidate relative costs and benefits of torpor to different groups within a population. We measured skin temperatures of 46 adult Rafinesque's big‐eared bats (Corynorhinus rafinesquii) to evaluate thermoregulatory strategies of a heterothermic small mammal during the reproductive season. We compared daily average and minimum skin temperatures as well as the frequency, duration, and depth of torpor bouts of sex and reproductive classes of bats inhabiting day‐roosts with different thermal characteristics. We evaluated roosts with microclimates colder (caves) and warmer (buildings) than ambient air temperatures, as well as roosts with intermediate conditions (trees and rock crevices). Using Akaike's information criterion (AIC), we found that different statistical models best predicted various characteristics of torpor bouts. While the type of day‐roost best predicted the average number of torpor bouts that bats used each day, current weather variables best predicted daily average and minimum skin temperatures of bats, and reproductive condition best predicted average torpor bout depth and the average amount of time spent torpid each day by bats. Finding that different models best explain varying aspects of heterothermy illustrates the importance of torpor to both reproductive and nonreproductive small mammals and emphasizes the multifaceted nature of heterothermy and the need to collect data on numerous heterothermic response variables within an ecophysiological context.     

Influence of undernutrition in the lactating cow on ovulation induction by serum gonadotropins (PMSG) during the post-partum period

Two experiments were carried out to assess the influence of undernutrition on postpartum ovulation in nursing Charolais cows after PMSG injection. In each experiment, the nursing cows were divided into 2 groups: one fed at a low nutritional level and the other at a normal nutritional level. In the first experiment, 19 animals were injected on post-partum days 15 and 30 with 600 IU of PMSG; in the second experiment, 34 received the same injection on post-partum day 54 after 9 days of priming with a Norgestomet implant. On post-partum day 15, only one cow in each group ovulated. At post-partum day 30, 1 out of 8 cows at the low nutritional plane ovulated vs 5 out of 9 at the normal nutritional plane (P less than 0.05). Likewise, on post-partum day 54, 5 out of 14 cows at the low nutritional plane ovulated vs 17 out of 17 at the normal nutritional level (P less than 0.05). Therefore, there is a time during the post-partum period when the nursing cow ovary does not respond to PMSG by ovulation. The length of this time is increased by undernutrition.     

Comparison of the amounts of hCG and PMSG to induce ovulation in 50% of the animals, mice, Syrian hamsters and rats]

On the day of diestrus, female mice, syrian hamsters and rats, showing regular 4-day estrous cycles, were injected with hCG or PMSG and were inspected for the presence of ovulation the following day. The dose level expected to cause an effect in 50% of the animals (ED50) was calculated using the Van der Waerden method. When hCG was injected into i. v., i. p. and s. c., the ED50 values per animal and per body weight (kg) in parenthesis were as follows; 0.2 (7.7), 0.3 (11.5) and 0.7 (26.9) I. U. for mice, 1.0 (9.5), 1.8 (17.1) and 2.6 (24.8) I. U. for syrian hamsters and 1.3 (4.6), 3.5 (12.3) and 7.5 (26.3) I. U. for rats, respectively. In PMSG study, the ED50 values per animal and per body weight (kg) in parenthesis were as follows: i. v., 0.8 (30.8); i. p., 2.0 (76.9); s. c., 2.8 (107.7) I. U. for mice, i. v., 3.6 (34.3); i. p., 8.0 (76.2); s. c., 13.2 (125.7) I. U. for syrian hamsters and i. v., 6.0 (76.8); i. p., 20.8 (73.0); s. c., 76.8 (269.5) I. U. for rats, respectively. From these results, the intravenous ED50 value was lower than other routes in three rodents with hCG or PMSG. In all injection routes, the ED50 value for mouse was lower than others. However, there were not significant differences in the ED50 values per body weight (kg) among three rodents. In particular, subcutaneous ED50 of hCG and intraperitoneal ED50 of PMSG were almost same values among three rodents, respectively. Given that the ED50 value per body weight (kg) in one of three rodents is determined, its value may be possible to be extrapolated to remaining two rodents.