Cyclic Hamster Research Articles

The Excitatory Effects of GABA within the Suprachiasmatic Nucleus: Regulation of Na-K-2Cl Cotransporters (NKCCs) by Environmental Lighting Conditions.

The suprachiasmatic nucleus (SCN) contains a pacemaker that generates circadian rhythms and entrains them with the 24-h light-dark cycle (LD). The SCN is composed of 16,000 to 20,000 heterogeneous neurons in bilaterally paired nuclei. γ-amino butyric acid (GABA) is the primary neurochemical signal within the SCN and plays a key role in regulating circadian function. While GABA is the primary inhibitory neurotransmitter in the brain, there is now evidence that GABA can also exert excitatory effects in the adult brain. Cation chloride cotransporters determine the effects of GABA on chloride equilibrium, thereby determining whether GABA produces hyperpolarizing or depolarizing actions following activation of GABAA receptors. The activity of Na-K-2Cl cotransporter1 (NKCC1), the most prevalent chloride influx cotransporter isoform in the brain, plays a critical role in determining whether GABA has depolarizing effects. In the present study, we tested the hypothesis that NKCC1 protein expression in the SCN is regulated by environmental lighting and displays daily and circadian changes in the intact circadian system of the Syrian hamster. In hamsters housed in constant light (LL), the overall NKCC1 immunoreactivity (NKCC1-ir) in the SCN was significantly greater than in hamsters housed in LD or constant darkness (DD), although NKCC1 protein levels in the SCN were not different between hamsters housed in LD and DD. In hamsters housed in LD cycles, no differences in NKCC1-ir within the SCN were observed over the 24-h cycle. NKCC1 protein in the SCN was found to vary significantly over the circadian cycle in hamsters housed in free-running conditions. Overall, NKCC1 protein was greater in the ventral SCN than in the dorsal SCN, although no significant differences were observed across lighting conditions or time of day in either subregion. These data support the hypothesis that NKCC1 protein expression can be regulated by environmental lighting and circadian mechanisms within the SCN.

Steroidogenic Pathway Proteins Change with Estrous Cycle and Photoperiod, but Are Not Affected by Inhibition of Matrix Metalloproteinases in Siberian Hamster Ovaries.

Restoration of ovarian function as a result of photostimulation corresponds with differential regulation of a family of proteases called matrix metalloproteinases (MMPs) in Siberian hamsters. Blocking MMP action with the in vivo inhibitor GM6001 impedes recrudescence: unlike vehicle treated controls, photostimulation of regressed animals treated with GM6001 fails to increase antral follicle numbers, ovulation, and plasma estradiol concentrations. Because normal recrudescence requires a return of estradiol synthesis and because inhibiting MMPs prevented the normal increase in plasma estradiol, we hypothesized that MMPs may be important in restoring steroidogenesis during photostimulated recrudescence. To test this hypothesis, we examined potential changes in steroidogenic enzymes 3β hydroxysteoid dehydrogenase (3βHSD), 17β hydroxysteoid dehydrogenase (17βHSD), aromatase, as well as estrogen receptor α in: stages of normal cycling hamsters (proestrus-P, estrus-E, diestrus I-DI, diestrus II-DII), hamsters exposed to long-days (LD; 16L:8D), regressed hamsters exposed to short days (SD; 8L:16D) for 14 wks, and regressed hamsters transferred to LD for 2 wks to stimulate recrudescence (PT). Hamsters in LD and PT groups were administered vehicle, IP GM6001, or no treatment daily for 2 wks. Immunoreactivity for 3β HSD protein was observed throughout the estrous cycle, with declines in extent, but not intensity of staining occurring at DII as compared to P, E, and DI (P < 0.05). No changes in 3βHSD immunostaining were noted in LD, SD, or PT ovaries, regardless of treatment with vehicle or GM6001 (P > 0.05). Both extent and intensity of 17β HSD immunostaining declined in DII as compared to other estrous cycle stages (P < 0.05). No changes in 17βHSD immunostaining were noted among LD ovaries, regardless of treatment; however, SD photoperiod decreased extent of immunoreactivity as compared to all groups regardless of treatment (P < 0.05). Aromatase immunostaining was noted throughout the estrous cycle, with declines in intensity and extent in DII as compared to other stages (P < 0.05). Aromatase was present in LD ovaries, and administration of vehicle or GM6001 did not alter intensity/extent of immunostaining (P > 0.05). In contrast, extent of aromatase immunoreactivity declined with SD exposure as compared to all other photoperiod groups (P < 0.05), although administration of vehicle or GM6001 did not affect a return of aromatase immunostaining in PT ovaries (P > 0.05). Finally, extent of immunostaining for ERα peaked in P as compared to DII (P < 0.05). Immunoreactivity for ER was observed in LD at low levels, regardless of treatment. In contrast to steroidogenic enzymes, intensity and extent of ER immunostaining peaked in SD, although administration of vehicle or GM6001 did not alter immunodetection. Our findings confirm and extend the cyclic nature of steroidogenic proteins, with most peaking in the active stages of the estrous cycle. Additionally, the significant decline in plasma estradiol that occurs with SD exposure is concomitant with decreases in later enzymes (17βHSD, aromatase) of steroidogenesis. Finally, although MMPs influence photostimulated return of ovarian function, they do not directly affect the return of ovarian steroidogenesis, as inhibiting MMP action did not affect these steroidogenic enzymes in either cycling LD females, or females undergoing recrudescence. Research supported by NIH 1SC3GM089611 (K.A.Y.) and CSUPERB-Howell Award (J.J.S.). (poster)

Differential ovarian expression of KiSS‐1 and GPR‐54 during the estrous cycle and photoperiod induced recrudescence in Siberian hamsters (Phodopus sungorus)

Kisspeptins, coded by the KiSS-1 gene, regulate aspects of the reproductive axis by stimulating GnRH release via the G protein coupled receptor, GPR54. Recent reports show that KiSS/GPR54 may be key mediators in photoperiod-controlled reproduction in seasonal breeders, and that KiSS-1/GPR54 are expressed in the hypothalamus, ovaries, placenta, and pancreas. This study examined the expression of KiSS-1/GPR54 mRNA and protein in ovaries of Siberian hamsters (Phodopus sungorus). Ovaries from cycling hamsters were collected during proestrus (P), estrus (E), diestrus I (DI), and diestrus II (DII). To examine KiSS-1/GPR54 during stimulated recrudescence, additional hamsters were maintained either in long day (LD 16L:8D, control) or short day (SD 8L:16D) for 14 weeks and then transferred to LD for 0-8 weeks. Staining of KiSS-1/GPR54 protein was detected by immunohistochemistry in steroidogenic cells of pre-antral and antral follicles, and corpora lutea. Immunostaining peaked in P and E, but decreased in the diestrus stages (P < 0.05). In recrudescing ovaries, KiSS-1/GPR54 immunostaining was low after 14 weeks of SD exposure (post-transfer [PT] week 0), and increased during the early weeks of recrudescence. Expression of KiSS-1/GPR54 mRNA was low with short day exposure, but increased during recrudescence and was higher at PT week 8 as compared to PT weeks 0 and 2 (P < 0.05). The elevated KiSS-1/GPR54 expression during P and E suggests a potential role in ovulation in Siberian hamsters. Transient increases in KiSS-1/GPR54 expression following LD stimulation are also suggestive of possible involvement in ovulation and/or restoration of ovarian function.

The Effects of Prolactin and Gonadotropin on Luteal Function and Morphology in the Cyclic Golden Hamster

The present study was conducted to evaluate the endocrinological effects of the pituitary on luteal maintenance and regression in the cyclic golden hamster (Mesocritus auratus). After hypophysectomy (Hypox) at 0900 h on day 1 of the estrous cycle (the day of ovulation), the animals received injection of prolactin (PRL) or PRL plus equine chorionic gonadotropin (eCG). They were decapitated at 1500 h on day 3 of the cycle, and trunk blood was collected for measurement of progesterone (P4). Corpora lutea (CLs) were dissected from one ovary for DNA ladder detection by electrophoresis, determination of DNA fragmentation ratio by fluorometric measurement method and measurement of P4. The other ovary was used for histological observation. After the Hypox, the daily injection of 1 mg ovine PRL restrained the DNA fragmentation ratio and number of apoptotic cell in the CLs. The PRL treatment maintained the luteal morphology and increased the luteal P4 concentration, but not in the plasma P4 concentration. In addition to PRL, injection of 2 IU eCG after the Hypox also restrained the DNA fragmentation ratio and number of apoptotic cells in the CLs to the level of a pregnant animal. The PRL plus eCG treatment maintained the luteal morphology in the same manner as the PRL only treatment and increased not only the luteal but also the plasma P4 concentration. These results suggest that PRL restrains luteal apoptosis and maintains luteal morphology and that the combination of PRL and eCG restrains not only structural but also functional luteal regression in the cyclic hamster.

Follicle stimulating hormone-induced DNA synthesis in the granulosa cells of hamster preantral follicles involves activation of cyclin-dependent kinase-4 rather than cyclin d2 synthesis.

Although cyclin D2 mRNA synthesis precedes gonadotropin-induced DNA synthesis in quiescent granulosa cells in culture, it is unclear whether a similar mechanism exists for the granulosa cells of growing preantral follicles in cyclic animals. The objective was to evaluate whether the synthesis of cyclin D2 protein was a prerequisite for FSH-induced DNA synthesis in the granulosa cells of intact preantral follicles of cyclic hamsters. Preantral follicles from cyclic hamsters were cultured in the presence or absence of FSH, and cell cycle parameters were examined. FSH stimulated cyclin-dependent kinase (CDK)-4 activity by 2 h and DNA synthesis by 4 h without altering the levels of cyclin D2 in the granulosa cells. The FSH effect was mimicked by epidermal growth factor administered in vivo. Although FSH increased the levels of cyclin D2 mRNA, it also stimulated the degradation of cyclin D2 as well as p27(Kip1) and p19(INK4) proteins. FSH activation of CDK4 was mediated by cAMP and ERK-1/2. In contrast to granulosa cells in intact follicles, FSH or cAMP significantly increased cyclin D2 protein levels in cultured granulosa cells but failed to induce DNA synthesis. Collectively, these data suggest that granulosa cells of preantral follicles, which are destined to enter the S phase during the estrous cycle, contain necessary amounts of cyclin D2 and other G1 phase components. FSH stimulation results in the formation and activation of the cyclin D2/CDK4 complex leading to DNA synthesis. This mechanism may be necessary for rapid movement of follicles from preantral to antral stages during the short duration of the murine estrous cycle.

Changes in expression of inhibin subunits in the cyclic golden hamster (Mesocricetus auratus) and the regulation of inhibin alpha subunit production by luteinizing hormone.

In the present study, changes in localization of each inhibin subunit in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization in inhibin alpha subunit in the ovary was also investigated. Inhibin alpha subunit was localized in granulosa cells of various stages of follicles throughout the estrous cycle. Inhibin alpha subunit was also present in numerous interstitial cells on days 1 and 2 (day 1 = day of ovulation), but the number of positive interstitial cells was fewer on days 3 and almost disappeared on day 4 of the estrous cycle. Newly formed luteal cells were also positive for inhibin alpha subunit on days 1 and 2. On the other hand, positive reactions for inhibin beta A and beta B subunits were only present in the granulosa cells of healthy antral follicles. However, a positive reaction for inhibin beta B subunit in peripheral mural granulosa cells disappeared on days 3 and 4 of the estrous cycle. Treatment with LHRH-AS at 1100 h on day 4 completely blocked the luteinizing hormone (LH) surge and ovulation, although relatively high concentrations of plasma follicle-stimulating hormone (FSH) were maintained throughout the experiment. There were few positive reactions for inhibin alpha subunit in theca and interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. These results suggest that the major source of dimeric inhibin in the cyclic hamster was granulosa cells of healthy antral follicles. Different distribution pattern of inhibin beta A from beta B subunits in large antral follicles on days 3 and 4 of the estrous cycle suggests different secretion patterns of inhibin A from B on these days. Furthermore, the LH surge may be an important factor to induce production of inhibin alpha subunit in interstitial cells of the cyclic hamster.

The Fate of Corpora Lutea in the Cyclic Golden Hamster

Structural luteolysis shows striking interspecies differences. Morphological changes in the corpus luteum (CL) of the cyclic hamster have been studied alongside the potential involvement of known luteolytic hormones. Ovaries from intact Syrian golden hamsters killed at 1100 h on days 1 and 2 and at 1100 and 1700 h on days 3 and 4 of the estrous cycle were dissected for histological study. The day of ovulation, the day of estrus, was arbitrarily designated day 1 of the estrous cycle. Steroidogenic cells in the CL were scarcely luteinized on day 1 and reached full luteinization on day 2. On the morning of day 3, initial regressive changes (accumulation of lipid droplets, invasion by neutrophils, and accumulation of phagocytic cells) were observed. These regressive changes increased progressively and apoptotic cells as well as phagocytic cells containing phagocytized apoptotic cells were abundant on the evening of day 3. On the morning of day 4, apoptotic cells/bodies and phagocytic cells containing phagocytized material were extremely abundant throughout the CL. However, steroidogenic cells with intact nuclei and well-preserved blood vessels were also found. Surviving cells in the CL showed progressive morphological changes. These cells showed morphological features intermediate between luteal and interstitial cells in the evening of day 4 and were virtually indistinguishable from interstitial cells on day 1 of the following cycle. Additional animals were injected at 1100 h on day 2 with: (a) the dopaminergic agonist CB154 (0.4 mg) to block prolactin secretion, (b) the anti-estrogen LY117018 (1.6 mg) or the anti-androgen Flutamide (3 mg) to block estrogen or androgen receptors, respectively, and (c) progesterone (2 mg) to prevent the fall in serum progesterone concentrations. Ovaries from these animals were collected at 1700 h on day 3 and at 1000 h on day 4. The luteolytic process was not affected by any treatment. These data indicate that, in contrast to its close relatives (e.g., the rat), structural luteolysis in the hamster is independent of the apoptotic inducing luteolytic hormones. In addition, differences in the cellular mechanisms responsible for CL elimination were also present. In the hamster, part of the luteal cells do not undergo apoptosis and seemed to progress through another developmental path giving rise to interstitial-like cells.

Can Pseudo Entrainment Explain the Synchrony of Estrous Cycles among Golden Hamsters (Mesocricetus auratus)?

Synchrony among golden hamsters is often cited in the menstrual-cycle synchrony literature and has recently become a paradigm for explaining menstrual synchrony in humans (L. Weller, A. Weller, and S. Roizman (1999), J. Comp. Psychol. 113, 261–268). It has also stimulated further research on synchrony in Djungarian hamsters, for which no evidence of synchrony was found (G. E. Erb, H. E. Edwards, K. L. Jenkins, L. C. Mucklow, and K. E. Wynne-Edwards (1993), Physiol. Behav. 54, 955–959). The case for synchrony in the golden hamster is reexamined in this paper. It is demonstrated, with the help of computer simulation experiments, that the experimental method used by G. Handelmann, R. Ravizza, and W. J. Ray (1980, Horm. Behav. 14, 107–115) for detecting synchrony has a critical flaw. It does not distinguish synchrony that can occur by chance (pseudo entrainment) from synchrony due to a process of entrainment. It is suggested that the apparent entrainment of estrous cycles in hamsters and the role of social dominance may be due to the stress caused by moving animals to different rooms or grouping them. Thus, because we cannot reject the possibility of pseudo entrainment as an explanation for these results, it must be concluded that there is no evidence that golden hamsters synchronize their estrous cycles. Finally, an approach is briefly outlined for testing synchrony in golden hamsters.

Regulations of gonadotropin secretion by circulating inhibin, estradiol, and progesterone in cyclic hamsters.

The physiological importance of gonadal hormones in feedback control of gonadotropin secretion during the estrous cycle in golden hamsters was investigated with immunoneutralization methods. Anti-inhibin serum (inhibin-AS) treatment always induced a drastic increase in follicle-stimulating hormone (FSH) secretion and occasionally raised luteinizing hormone (LH) secretion. Anti-estradiol-17beta serum (estradiol-AS) treatment increased LH secretion typically. Although estradiol-AS elevated FSH secretion occasionally, the elevation was much less than that by inhibin-AS. Plasma FSH reached ovariectomized levels by a synergistic effect of both antisera. Elevated plasma LH with both antisera was much less pronounced than in ovariectomized animals. Plasma LH increased dramatically to the levels in the ovariectomized group when antibody against progesterone (progesterone-AB) was given together with inhibin-AS and estradiol-AS, although progesterone-monoclonal antibody alone did not alter plasma gonadotropin levels. These results indicate that in hamsters FSH secretion is mainly regulated by inhibin and LH secretion is regulated by estradiol-17beta and progesterone during the estrous cycle.

Melatonin directly suppresses steroid production by preovulatory follicles in the cyclic hamster.

The purpose of these studies was to investigate the effects of melatonin on the production of steroids (progesterone, testosterone, and estradiol) and cAMP by preovulatory follicles and to examine changes in melatonin concentrations in the ovary during the estrous cycle. Adult cyclic hamsters were used in this study. Melatonin concentrations in the ovary, pineal gland, and serum were measured at mid-light and mid-dark during the estrous cycle. Effects of melatonin on steroidogenesis by preovulatory follicles, thecae, and granulosa cells were examined, and its effect on cAMP production by preovulatory follicles was also investigated. Melatonin (0.1-10 ng/ml) had no effect on steroid production in the absence of hCG, but melatonin decreased progesterone and estradiol production by preovulatory follicles in a dose-dependent and time-dependent manner in the presence of hCG (100 mIU/ml). The target of melatonin was thecae but not granulosa cells, and melatonin significantly reduced cAMP production by preovulatory follicles. Melatonin concentrations in the ovary showed a similar phasic variation with high levels during mid-dark and low during mid-light, as in the pineal gland and serum. These results show that the ovarian melatonin levels also exhibit a circadian rhythm and suggest that the high melatonin milieu in the ovary may induce gonadal regression in the cyclic hamster.

Variations of prostaglandin E 2 receptors in hamster's ovary and endometrium during estrous cycle

The purpose of this study was to determine concurrent ovarian and endometrial prostaglandin E 2 (PGE 2) receptor concentrations throughout the hamster estrous cycle. The effect of progesterone (P 4) on PGE 2 receptor in these two tissues was also investigated during in vitro culture. Estrous cycles of mature female hamsters were monitored according to the appearance of the vaginal discharge on cycle day 1 (D1). Ovaries and uteri were removed from cyclic hamsters at 10:00 a.m. on each day of the cycle and at 6:00 p.m. on D4 (proestrus). Ovarian and endometrial cell membranes were collected and assayed for the specific PGE 2 binding by Scatchard plot analysis, using seven different concentrations of 3H-PGE 2 (0.72–9.1 nM) with or without the presence of unlabelled PGE 2 (9.1 μM). Ovarian and endometrial tissues of the cyclic hamsters were shown to contain a saturable, specific binding site with K d =4.69 ±0.55, and 5.7 ± 0.4 nM for ovary and endometrium, respectively. Relative binding activity of PGE 1 for the PGE 2 binding site was about 28%. PGF 2α and PGA 1 did not compete for the PGE 2 binding site. In ovaries, the PGE 2 receptor levels started to increase sharply in the evening of D4 and reached maximum in the morning of D1. A precipitous drop of PGE 2 receptor was observed on D2 followed by gradual decreases on D3 and D4. The PGE 2 receptor concentrations in endometrium were the lowest in the morning of D4, and increased thereafter until a maximal level was reached on D2. Progesterone (10 nM) augmented PGE 2 receptors in ovarian but not in endometrial tissue during 24-h in vitro incubation.

Apoptosis during spontaneous luteolysis in the cyclic golden hamster: biochemical and morphological evidence.

The corpora lutea (CL) of the cyclic hamster are destroyed between Days 2 and 3 of the 4-day estrous cycle so that only one set is ever present (Day 1 = estrus, Day 4 = proestrus). The possibility that luteal cell death in the cyclic hamster is attributable to apoptosis was explored. The earliest histological signs of structural luteolysis were detected at 0600 h of Day 3 as evidenced by a few scattered luteal cells displaying the characteristic morphology of apoptotic cells and by a massive infiltration of neutrophils. The peaks of neutrophil influx and luteal apoptosis were reached on Day 3, 1200 h, and Day 3, 2400 h, respectively. Thus, the increase in neutrophils occurs before the major onset of luteolysis. By Day 3, 2400 h, the CL had already shrunken one third by weight, and they virtually vanished by the next Day 1. Apoptosis ultimately destroyed luteal endothelial cells, luteal cells, and neutrophils. Electrophoretic analysis of low-molecular weight DNA in luteal cell lysates revealed a definite ladder pattern of oligonucleosomal-length DNA fragments--characteristic of apoptosis--on Day 3 beginning at 1200 h. The pattern was not detectable in CL collected on Day 2. Comparing Day 3 CL collected at 0900-1200 h with those at 1500-1800 h showed that only the latter group exhibited inter-nucleosomal cleavage activity. The minimal number of CL on Day 3, 1500 h, needed to demonstrate DNA laddering was six. In summary, the electrophoretic separation of oligonucleosomal fragments and histology indicated that apoptosis occurs during spontaneous luteal regression on Day 3 of the hamster cycle. The initiation of apoptosis is not apparent until several hours after the onset of functional luteolysis. The rapidity with which apoptosis eliminates the CL over a very precise time schedule makes the cyclic hamster an ideal model to analyze the factors involved in structural luteolysis.

Effects of the fructose analog, 2,5-anhydro-D-mannitol, on food intake and estrous cyclicity in Syrian hamsters.

Hyperphagia and anovulation are both triggered by prior food deprivation or other treatments that decrease intracellular availability of metabolic fuels in most species studied. Syrian hamsters fail to show compensatory hyperphagia, but do show anestrus in response to these energetic challenges. In the present experiments, we examined food intake, plasma glucose levels, and estrous cyclicity in Syrian hamsters in response to 2,5-anhydro-D-mannitol (2,5-AM), a fructose analog that is thought to trigger eating in rats by depleting intracellular levels of ATP. In experiment 1, female estrous cycling hamsters were treated with 100, 200, 400, or 800 mg/kg 2,5-AM or the vehicle by intraperitoneal injection. Food intake was measured 1, 2, 4, 8, and 24 h after treatment. There were no statistically significant increases in food intake in response to any dose of 2,5-AM. In experiment 2, blood samples were drawn at 0, 1, 3, 5, 7, and 25 h after hamsters were treated with 0 or 400 mg/kg 2,5-AM. 2,5-AM treatment resulted in a mild but significant decrease in plasma glucose levels similar to those seen in 2,5-AM-treated rats, suggesting that 2,5-AM has similar effects on fuel metabolism in rats and hamsters. In experiment 3, hamsters received 2,5-AM, 2,5-AM plus the fatty acid oxidation inhibitor methyl palmoxirate, or vehicle every 6 h over the first 48 h of the estrous cycle and were tested for indexes of estrous cyclicity at the end of the cycle. All hamsters showed normal estrous cycles, regardless of treatment. If 2,5-AM has similar metabolic consequences in rats and hamsters, the present results suggest that decreased intracellular levels of ATP and mild hypoglycemia do not increase food intake or inhibit estrous cyclicity in Syrian hamsters.

Temporal Changes in Inhibin, Steroid Hormones, and Steroidogenic Enzymes during Induced Follicular Atresia in the Hypophysectomized Cyclic Hamster1

The time course for loss of ability of Graafian follicles to secrete inhibin and estradiol was investigated during induced follicular atresia. Cyclic hamsters were hypophysectomized on Day 1 (estrus) and injected s.c. with 30 IU eCG. Thereafter, these animals were given a single i.p. injection of antiserum to eCG on the morning of Day 4 to induce follicular atresia in a rapid and predictable manner. A drastic fall in plasma levels of estradiol and testosterone was noted within 1 h, whereas relatively high levels of plasma inhibin were maintained until 12 h, followed by an abrupt decline by 24 h. The first histological signs of pyknosis in granulosa cells appeared by 4 h, and breakdown of the mural granulosa layer was observed in most follicles by 8-12 h after immunoneutralization of circulating eCG. According to immunohistochemical analysis, inhibin activity was unchanged in granulosa cells at 12 h followed by a slight decline by 24 h, whereas positive reaction for aromatase in these cells rapidly declined by 8 h. Immunoreactivity of 17alpha-hydroxylase/C17,20-lyase (CYP 17) was also reduced in theca cells by 8 h. These results indicate that granulosa cells continue to secrete inhibin during the process of follicular atresia, although these cells quickly shut off the secretion of estradiol, and theca cells shut off the secretion of testosterone. The present results indicate as well that a rapid decline of estradiol and testosterone in plasma is an early sign of atresia in antral follicles. These results, therefore, suggest that the loss of enzymatic activity of aromatase in granulosa cells and CYP 17 in theca cells is a part of the process of follicular atresia.

Low Plasma Estradiol is Required for the Expression of Daily Increase in Plasma Gonadotropins in the Lactating Golden Hamster (Mesocricetus auratus).

Mechanisms responsible for a daily increase in plasma gonadotropins during lactation of golden hamsters (Mesocricetus auratus) were investigated. A daily afternoon (1700 h) increase in plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH) and progesterone was observed until at least day 15 of lactation (day 0 of lactation = day of parturition). On the other hand, plasma estradiol did not show any significant change at 1100, 1700 and 2300 h. Such increases occur in hamsters nursing 8 pups. In hamsters nursing 2 pups, however, only a daily increase in LH was noted on day 5 of lactation and the diurnal rhythm of LH was difficult to see because of the increase in basal levels of LH on day 10 and 15 of lactation. The diurnal increases in plasma LH and FSH disappeared within 2 days after removal of the litter on day 10 of lactation and by day 14 of lactation (4 days after litter removal) most animals had resumed ovulating. Ovariectomy on day 2 of lactation did not affect the profile of plasma LH on subsequent days. In contrast, the basal levels of plasma FSH increased, suggesting the absence of an inhibitory ovarian signal (probably inhibin). A negative relationship existed between plasma estradiol and the daily surges of LH and FSH at 1700 h; the daily rise in gonadotropins occurred when plasma levels of estradiol were low. The afternoon increases in plasma LH and FSH were prevented by treatment with physiological levels of estradiol, comparable to those observed in the cyclic hamster. Antiserum against LHRH injected at 1100 h on day 10 of lactation completely suppressed the daily rise of plasma LH and FSH. These results indicate that during lactation low levels of estradiol are required for the expression of diurnal increase in plasma LH and FSH in the hamster. Moreover, the low levels of estradiol results in the daily release of LHRH.

Induction of superovulation by immunoneutralization of endogenous inhibin through the increase in the secretion of follicle-stimulating hormone in the cyclic golden hamster.

The present study was conducted to study the effect of immunoneutralization against endogenous inhibin on FSH, LH, oestradiol-17 beta and progesterone secretion and to investigate the effect of removal of endogenous inhibin on subsequent follicular development in the hamster. After treatment with anti-inhibin serum (inhibin-AS) at 1100 h on day 2 of the oestrous cycle (day 1 = day of ovulation), a marked increase in plasma levels of FSH and a slight increase in plasma levels of LH were noted and pituitary contents of FSH, but not LH, were also increased. In the group treated with inhibin-AS, superovulation occurred on day 1 of the following cycle. Plasma levels of oestradiol-17 beta markedly increased with the increase in the number of ovulations induced by human chorionic gonadotrophin (hCG) as compared with those in control animals. In the second cycle, plasma concentrations and pituitary contents of FSH in the animals given 200 microliters inhibin-AS still showed high values as compared with those in the animals treated with control serum, although superovulation did not occur on day 1 of the third cycle. Plasma concentrations and pituitary contents of LH in the hamster given 200 microliters inhibin-AS tended to decrease as compared with those in control animals during the second cycle. Plasma concentrations of oestradiol-17 beta in the animals treated with 200 microliters inhibin-AS changed in a similar way to controls. A marked increase in plasma concentrations of progesterone was noted on days 1 and 2 of the second cycle in the group receiving inhibin-AS. The twice daily injection of 1 IU hCG during the second cycle to the animals given 200 microliters inhibin-AS induced superovulation on day 1 of the third cycle. These results indicate that circulating inhibin may be an important indicator of the number of developing follicles and may maintain the species-specific number of developing follicles through suppression of FSH secretion in the cyclic hamster. They also suggest that high levels of inhibin slightly suppress plasma levels of LH, indicating that plasma LH may also regulate follicular development in the cyclic hamster.

Changes in interleukin-1 production of peritoneal macrophages during estrous cycle in golden hamsters.

The present study demonstrated the change in interleukin-1 (IL-1) production of peritoneal macrophages during the estrous cycle in golden hamsters and discussed its possible roles in ovarian function. Macrophages were collected from the peritoneal cavity at 0900 h on various days of the estrous cycle and incubated for 6 h in the presence of ovine pituitary LH (500 ng/ml). The IL-1 concentration in the media was measured by bioassay with the A375S2 human melanoma cell line. The number of macrophages significantly (P < 0.01) increased on estrus and proestrus compared with diestrus 1 or diestrus 2. LH-induced production of IL-1 was also greater (P < 0.01) on proestrus (292 +/- 36 pg/10(6) cells/ ml) and estrus (222 +/- 30 pg/10(6) cells/ml) than on diestrus 1 (34 +/- 15 pg/10(6) cells/ml) or diestrus 2 (117 +/- 16 pg/10(6) cells/ml). To clarify the factor inducing the changes in peritoneal macrophages, hamsters were ovariectomized on diestrus 1, and 3 weeks later the animals were treated with s.c. injections of progesterone (200 micrograms/day), testosterone (100 micrograms/day), estradiol (10 micrograms/day) or sesame oil for three days. The hamsters were killed 24 h after the last injection, and the number and IL-1 producing capacity of macrophages were determined. The number of macrophages and their response to LH to produce IL-1 were increased significantly (P < 0.01) by estradiol treatment but not by progesterone or testosterone treatment. It was concluded that the peritoneal macrophages became more sensitive to LH to produce IL-1 on proestrus and estrus in cyclic hamsters, and that these changes in macrophages, probably induced by estradiol, would play important roles in ovarian function.

Follicular selection in the cyclic hamster: a 1960 model.

Follicular selection in the cyclic hamster: a 1960 model.

Possible Role of Platelet-Activating Factor in Embryonic Signaling during Oviductal Transport in the Hamster1

Hamster embryos enter the uterus in pregnant females nearly one day earlier than unfertilized eggs in cycling females. The hypothesis that a substance derived from eicosanoids is released by the embryos, but not by oocytes, to hasten their transport to the uterus was tested by examining the effects of indomethacin, nordihydroguaiaretic acid (NDGA), platelet-activating factor (PAF), and PAF antagonists on egg transport in the hamster. Administration of indomethacin had no effect on embryo transport, whereas administration of NDGA delayed the transport of eggs to the uterus in pregnant but not in cycling hamsters. The PAF antagonists TCV-309 and BN-52021 delayed significantly the transport of eggs to the uterus in pregnant animals, but not in cycling animals; i.e., they retarded the passage of embryos but not of oocytes to the uterus. Administration of PAF to cycling hamsters hastened the oviductal transport of ova. These data suggest that, in the hamster, the earlier passage of embryos to the uterus as compared to oocytes is mediated by PAF. Thrombocytopenia was detected in early-pregnant hamsters, and PAF-like activity was detected in spent media of two-cell through morula stage hamster embryos. These results suggest that preimplantation hamster embryos produce PAF-like activity that mediates embryonic signaling to the oviduct as well as pregnancy-associated thrombocytopenia.

Antisense epidermal growth factor oligodeoxynucleotides inhibit follicle-stimulating hormone-induced in vitro DNA and progesterone synthesis in hamster preantral follicles.

Whether FSH-induced follicular DNA synthesis in the hamster involves epidermal growth factor (EGF) message induction and translation was evaluated using antisense EGF oligodeoxynucleotides. The 15 base pair antisense oligomers synthesized according to mouse EGF cDNA sequence downstream from the 5'-ATG start site hybridized with a 4.4-kilobase message of mouse submaxillary glands and with 4.0 kilobases of mRNA of hamster submaxillary glands, kidney, and ovaries. Preantral follicles at stages 1-7 were enzymatically and mechanically isolated from adult, cyclic hamsters on day 4:0900 h (day 1 = estrus), and follicles at stages 1-5 were isolated from hamsters hypophysectomized for 10 days. Follicles were preexposed to either antisense or sense EGF thio-oligomers for 1, 12, or 24 h and then cultured for an additional 24 h in the absence or presence of ovine-FSH (100 ng/ml) or recombinant murine EGF (50 ng/ml), and 1 microCi/ml [3H]thymidine. Follicular [3H]thymidine incorporation and EGF and steroid (progesterone, androstenedione, and 17 beta-estradiol) production were monitored to assess the molecular mechanism of FSH regulation of follicular development. Antisense oligomers significantly inhibited FSH-induced follicular DNA synthesis and progesterone but not androgen or estrogen production with a latency of 24 h. The oligomer inhibition of FSH action was effectively reversed by exogenous EGF. Antisense oligomers significantly inhibited follicular EGF synthesis in vitro. These results suggest that EGF message is expressed in the hamster ovary, and one of the molecular mechanisms of FSH regulation of hamster preantral folliculogenesis involves EGF mRNA induction and translation.(ABSTRACT TRUNCATED AT 250 WORDS)