Hypothalamic alterations and reproductive aging in female rats: evidence of altered luteinizing hormone-releasing hormone neuronal function

Prior to the age-related loss of regular estrous cycles, female rats exhibit an attenuated preovulatory LH surge, a sign that reproductive decline is imminent. Numerous studies have revealed an important role for the hypothalamus in aging of the reproductive axis in this species. Because LHRH represents the primary hypothalamic signal that regulates gonadotropin release, assessments of LHRH neuronal activity can provide a window into hypothalamic function during reproductive aging. Studies of the dynamic activity of LHRH neurons during times of enhanced secretion have revealed deficits in middle-aged females. Available data are consistent with a decline in LHRH synthesis, transport, and secretion in middle-aged females during times of increased demand for LHRH output. Moreover, the alterations noted in LHRH neuronal function could account, in part, for the attenuation and eventual loss of the preovulatory LH surge with age. Elements extrinsic to LHRH neurons undoubtedly contribute to the decline in the parameters of LHRH neuronal function observed in middle-aged females. Whether alterations intrinsic to LHRH neurons also play a role in the age-associated reduction in LHRH synthesis and secretion remains to be determined. Recent examinations of hormone profiles during the perimenopausal period suggest that a potential hypothalamic contribution to aging of the reproductive axis in women warrants further examination.     

Hypothalamic luteinizing hormone-releasing hormone (LHRH) and LH secretion in aging female rats

Age-related changes in hypothalamic luteinizing hormone-releasing hormone (LHRH) and luteinizing hormone (LH) secretion were studied in young (6 months), middle-aged (12 months) and old (18 months) female rats. The LHRH levels in the mid-hypothalamic area were higher in intact middle-aged and old females than in young ones. Additionally, there was no age difference in the hypothalamic LHRH levels in male rats. In order to clarify the significance of this age-related increase in female rats, we examined the effects of progesterone treatment in estrogen-primed ovariectomized young and old rats on the LHRH levels in the median eminence (ME) and on plasma LH levels. We found phasic changes in ME-LHRH and plasma LH levels in estrogen-primed rats following progesterone treatment in rats of both ages, but the progesterone-induced change in ME-LHRH levels tended to be delayed in old rats compared with young females. This delay may correspond to the delayed onset, slow and low magnitude of plasma LH increase in old females. The ME-LHRH levels were generally higher in old rats than in young rats. Nevertheless, we found that the increase in plasma LH in response to progesterone treatment in estrogen-primed ovariectomized females was smaller in old rats than young rats. These results suggest that the LHRH secretory mechanism changes with age in female rats. Such alterations may result in the accumulation of LHRH in the mid-hypothalamic area and an increase in ME-LHRH.     

Gonadal steroids and neuropeptide Y-opioid-LHRH axis: interactions and diversities

We report that the two classes of regulatory neuropeptides, neuropeptide Y (NPY) and endogenous opioid peptides (EOP), modulate luteinizing hormone (LH) release in diverse fashion in gonad-intact rats. Each neuropeptide acts at two loci, the hypothalamus and pituitary, to excite (NPY) or inhibit (EOP) LH release. At the hypothalamic level, NPY stimulates luteinizing hormone releasing hormone (LHRH) release, a response mediated by alpha 2-adrenoreceptors and amplified in the presence of adrenergic agonists. At the pituitary level, NPY acts in concert with LHRH to amplify the LH response. In contrast, EOP inhibit LHRH release by decreasing the supply of excitatory adrenergic signals in the vicinity of LHRH neurons in the preoptic-tuberal pathway, and at the pituitary level, they decrease LH release in response to LHRH. Further, the gonadal steroidal milieu facilitates NPY neurosecretion and postsynaptic expression of NPY in concert with adrenergic system; a similar clear-cut facilitatory effect of gonadal steroids on EOP secretion is not yet obvious. Our additional studies imply that the EOP system has the potential to increase sensitivity towards gonadal steroids and that to induce the preovulatory LH surge the neural clock may decrease the inhibitory EOP tone prior to the critical period on proestrus. This antecedent neural event allows the excitatory adrenergic and NPY signals to evoke LHRH secretion at a higher frequency approximating that seen in ovariectomized rats. Further studies are under way to delineate the steroid-induced subcellular events that integrate the action of these regulatory peptides in the control of the episodic LHRH secretion pattern which sustains basal and cyclic gonadotropin release in the rat.     

Neuroendocrine regulation of pulsatile luteinizing hormone secretion in elderly men

Leydig cell function is driven by LH, secreted in a pulsatile manner by the anterior pituitary in response to episodic discharge of hypothalamic LHRH into the pituitary portal circulation, under control of a yet to be defined neural mechanism, the "hypothalamic LHRH pulse generator". The normal aging process in elderly men is accompanied by a decline in Leydig cell function. Whereas primary testicular factors undoubtedly play an important role in the decrease of circulating (free) testosterone levels with age, recent studies demonstrated that aging also affects the central compartment of the neuroendocrine cascade. Hypothalamic alterations comprise changes in the regulation of the frequency of the LHRH pulse generator with an inappropriately low frequency relative to the prevailing androgen impregnation and opioid tone, and with an increased sensitivity to retardation of the LHRH pulse generator by androgens. As observed by some authors in basal conditions and by others after endocrine manipulations. LH pulse amplitude seems also to be reduced in elderly men as compared to young subjects. This is most probably the consequence of a reduction in the amount of LHRH released by the hypothalamus. Indeed, challenge of the gonadotropes with low, close to physiological doses of LHRH in young and elderly men reveals no alterations in pituitary responsiveness when looking at either the response for immunoreactive LH or bioactive LH. Deconvolution analysis on data obtained after low-dose LHRH suggests a markedly prolonged plasma half-life of LH in elderly men, a finding which may explain the paradoxical increase of mean LH levels in face of the reduced or unchanged frequency and amplitude of LH pulses.     

Diazepam: endocrine effects and hypothalamic binding sites in the developing male and female rat

The ontogeny of diazepam's endocrine effects in male and female rats, and of 3H-diazepam binding in the hypothalami of both sexes was studied. Diazepam inhibited basal prolactin levels in 38 day-old male rats and, if prolactin levels were stimulated by Haloperidol the inhibition occurred in 28 day-old males, indicating that the hypoprolactinemic effect of the drug could be evidenced earlier if prolactin titers were high. The prolactin inhibition in females did not reach statistical significance at any studied age. Diazepam significantly released LH only in male rats at 12 days, showing thus, a period of special sensitivity of LH release to the drug. Benzodiazepine-hypothalamic binding sites increased in number from birth to puberty, reaching a plateau at 20 days of age. No sexual differences or changes in affinity were found throughout the studied period. These results suggest that the maturation of diazepam's hypoprolactinemic effect could be partially related to the increase in hypothalamic binding sites, whereas the sexual differences observed in diazepam's endocrine actions could be due to sexual differentiation of endocrine control mechanisms.     

Further evidence that prolactin does not affect gonadotropin release at pituitary level

In a primary monolayer cell culture of the anterior pituitary from mature male rats the effects of exogenous rPrl (rPrl exog.) and endogenously secreted rPrl (rPrl endog.) on basal and LHRH stimulated LH secretion were investigated. In pilot studies basal Prl- and LH secretion as well as influence of various LHRH concentrations (10(-1)-10(+3) ng/ml) on Prl- and LH release were observed. The influence of exogenous rPrl was studied at various concentrations (50-500 ng/ml) and with preincubation periods of 2 hrs and 6 hrs before starting LHRH stimulation. The dopamine agonist bromocriptine and the dopamine antagonist sulpirid were preferentially used to prove physiologic function of the cell system presented. Basal LH secretion started after a delay of 3 hrs, whereas basal Prl secretion began immediately showing a linear rise for 9 hrs. LHRH stimulation resulted in a non-linear dose and time dependent LH secretion. LHRH showed no influence on endogenous Prl (rPrl endog.) secretion of the mammotroph cells. Exogenous Prl (rPrl exog.) did not affect spontaneous Prl release excluding ultra short loop inhibition in this cell system. Furthermore, exogenous Prl had no effect on either basal or LHRH stimulated LH secretion even after a preincubation period of up to 6 hrs and at concentrations generally observed for prolactin secreting tumors. Bromocriptine suppressed endogenous Prl release and did not affect LH secretion. Sulpirid had no influence on either Prl or LH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian steroidogenic responsiveness to exogenous gonadotropin stimulation in young and middle-aged female rats

Reproductive aging in the female rat is associated with gradual declines in LH secretion and ovarian progesterone (P) production. This study examined whether the influences of aging on P levels reflect decreased ovarian responsiveness to gonadotropin stimulation, as opposed to changes in gonadotropin release. Young and middle-aged regularly cyclic female rats received sodium pentobarbital to block endogenous proestrous luteinizing hormone (LH) surges, followed by administration of various doses of human chorionic gonadotropin (hCG). Similar treatments were performed in middle-aged acyclic persistent-estrous (PE) females. Injection of hCG resulted in equivalent plasma hCG levels in each treatment group. At the lowest hCG dose tested, a significant rise in plasma P levels was observed in middle-aged cyclic rats, but not in young cyclic or middle-aged PE females. This unexpected finding may reflect accelerated follicular development in middle-aged cyclic females, as suggested by a previous study. At the intermediate dose, young and middle-aged cyclic but not PE rats displayed significantly increased P in response to hCG. At the highest dose tested, all three groups of rats displayed increased P levels after hCG stimulation. However, P concentrations were significantly lower in middle-aged PE than regularly cyclic females. Northern and slot blot hybridization analyses revealed that ovarian mRNA levels for cytochrome P450 side-chain cleavage, the rate-limiting enzyme in P synthesis, were markedly reduced in PE rats following hCG stimulation. These findings indicate that ovarian responsiveness to gonadotropin stimulation is impaired in middle-aged PE, but not regularly cyclic rats, and suggest influences of cycle status on the biochemical and molecular mechanisms regulating ovarian steroid production. Furthermore, these findings reveal that attenuated P production in middle-aged proestrous rats is due to attenuated preovulatory LH surges, rather than decreased ovarian sensitivity to LH.     

In vitro release of hypothalamic corticoliberine in the rat: incubation of slices from the whole hypothalamus

An experimental system allowing both the incubation and rapid transfert of rat hypothalamic slices has been developed in order to approach the regulation of CRF secretion. The release of CRF has been quantified by a specific radioimmunoassay. Under basal conditions, immunoreactive CRF release reached an optimum of 96.2 +/- 10.4 pg/3 hypothalami/20 min. A depolarizing concentration of KCl (56 mM) or veratridine (50 microM) applied for 20 min. induced a 222 and 257% increase, respectively, in CRF release. The in vitro CRF values released under basal and stimulated conditions are comparable to those of other hypothalamic neuropeptides. Furthermore, in vitro CRF release from the hypothalamus is in the same order of magnitude as in vivo CRF secretion estimated by hypophysial portal blood collection or median eminence push-pull cannulation.     

Effect of time after castration on secretion of LHRH and LH in the ram

Hypophysial portal blood and peripheral blood were obtained from conscious, unrestrained rams to measure simultaneously the secretion of LHRH and LH in entire rams and rams which had been castrated for 2-15 days (short-term castration) and for 1-6 months (long-term castration). The apparatus for portal blood collection was surgically implanted using a transnasal trans-sphenoidal approach and, 4-5 days later, portal blood and peripheral blood were collected simultaneously at 10-min intervals for 8-9 h from 15 sheep. LHRH was clearly secreted in pulses in all three physiological conditions, but there were marked differences in pulse frequencies, which averaged 1 pulse/2-4 h in entire rams, 1 pulse/70 min in short-term castrated rams and 1 pulse/36 min in long-term castrated rams. In entire and short-term castrated animals, LH profiles were also clearly pulsatile and each LHRH pulse in hypophysial portal blood was associated with an LH pulse in the peripheral blood. In long-term castrated animals, LH pulses were not as well defined, because of the high basal levels and small pulse amplitudes, and the temporal relationship between LHRH and LH pulses was not always clear. These results demonstrate the pulsatile nature of LHRH secretion under the three physiological conditions and suggest that the irregular LH profiles characteristic of long-term castrates are due to an inability of the pituitary gland to transduce accurately the hypothalamic signal. The very high frequency of the LHRH pulses may be one of the major reasons for this, and is probably also responsible for the high rate of LH secretion in the long-term castrated animal.     

Sex steroids and the control of LHRH secretion

Gonadal steroids are important hormonal signals that regulate the activity of LHRH synthesizing and releasing neurons. Aside from a direct effect through the feedback mechanisms exerted at hypothalamic and/or anterior pituitary level, gonadal steroids may modify the rhythmic LHRH release by modulating other systems affecting LHRH neurons. 1. In ovariectomized E2-treated female rats, progesterone is able to evoke LHRH release from the perifused hypothalamus without affecting LH and FSH release. 2. Excitatory amino acids (EAA) and their related analogs (NMDA and kainate) are known to stimulate LH release in young rats. When tested in a perifusion system on hypothalamic and anterior pituitary tissues, they differentially stimulate the release of LHRH (NMDA) and of LH (KA); their effect on both structures is markedly reduced following orchidectomy. It appears that gonadal steroids might exert a facilitatory action on the neurosecretory activity of LHRH neurons as well as a modulatory influence on the effect of EAA.     

Age-related alterations in the stimulated release in vitro of catecholamines and luteinizing hormone-releasing hormone from the male rat hypothalamus

Using an in vitro perifusion system, the present study investigated the possibility that alterations in catecholamine and luteinizing hormone-releasing hormone (LHRH) secretion from the male rat mediobasal hypothalamus are present during the period of middle-age. The results indicate that, while tissue concentrations and baseline secretion of norepinephrine, dopamine and LHRH were similar between age groups, the patterns of dopamine and LHRH release in response to a series of depolarizing stimuli was different in the older animals. After all challenges, dopamine concentrations in the perifusate declined much more sharply for the middle-aged group, a finding that may be associated with a decrease with age in the pool of transmitter available for ready release. Also, tissue fragments from young adult rats were able to maintain the release of LHRH to a greater extent than tissue from the middle-aged animals, but only for the initial challenge period. The typical episodic pattern of LHRH release appeared to be disrupted in the older group following a second stimulus. It is possible that these age-related changes are early components of a dysruption in the hypothalamic mechanisms governing gonadotropin secretion.The research described in this article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade name or commercial products constitute endorsement or recommendation for use.     

Changes in populations of LHRH-immunopositive cell bodies following gonadectomy

One day after castration of male rats, plasma LH rose and the number of LHRH immunopositive neuronal perikarya decreased. As plasma LH continued to rise six days and three weeks post-castration, the number of LHRH immunopositive neurons also increased. The largest population of LHRH immunopositive neurons was detected three weeks post-castration and the cell group that showed the greatest increase was in the rostral preoptic area. In females, the largest population of LHRH immunopositive neurons was observed one day post-ovariectomy; at this time plasma LH levels were not significantly elevated above diestrous levels. Six days post-ovariectomy, LH levels were elevated and the number of LHRH immunopositive cells decreased. As LH levels continued to rise three weeks post-ovariectomy, the population increased in size. In males, primarily LHRH cells of the rostral preoptic area increased in in number; in females, the cell groups that increased were scattered over the diagonal band of Broca, preoptic and anterior hypothalamic areas. Although LHRH neurons demonstrated these variations following gonadectomy, there was no evidence of alteration(s) in molecular processing of precursor hormone.     

Effects of Superior Cervical Ganglionectomy and Melatonin Replacement on Intra-hypothalamic LHRH Content and Pulsatile Luteinizing Hormone Release in the Mink

Long-term effects of subcutaneous melatonin implants on intrahypothalamic LHRH content and on pulsatile luteinizing hormone release have been investigated in ganglionectomized male mink. Animals were submitted to bilateral removal of the superior cervical ganglion in mid-April. A preliminary study revealed that plasma LH concentrations remain at a basal level throughout the year following ganglionectomy. In a second experiment, one month after ganglionectomy and transfer from the natural photoperiod environment to short daylengths (LD 4:20), melatonin pellets were subcutaneously implanted to overcome deafferentation of the pineal. Progressive effects of treatment were studied 7 days, 15 days, and one, two and three months after insertion of the melatonin implants. The intra-hypothalamic LHRH content in ganglionectomized mink was at a basal level similar to that observed during seasonally sexual quiescence, or after exposure to inhibitory long days (LD 20:4). A significant and transient elevation in LHRH content was observed already after fifteen days, and also one month after insertion of melatonin implants. This resulted in mean values similar to those observed during the breeding season, or after exposure to stimulatory short days (LD 4:20). A decrease in hypothalamic LHRH content started after two months. No pattern of pulsatile LH secretion was recorded in ganglionectomized untreated mink. A significant increase in all parameters of pulsatile LH secretion was observed fifteen days after the elevation of LHRH content induced by melatonin treatment, and maximum values were reached after two months. Pituitary activity tended to decrease after three months, characterized in particular by a significant decrease in the mean frequency of LH pulses. In addition, the increase in pulsatile characteristics of LH release occurred two months before the peripheral renewal of testicular activity. Apparently, the reproductive endocrine function in ganglionectomized mink treated with melatonin implants is restored more rapidly at the hypothalamic level than at the pituitary or testicular levels.     

Effects of Superior Cervical Ganglionectomy and Melatonin Replacement on Intra-hypothalamic LHRH Content and Pulsatile Luteinizing Hormone Release in the Mink

Long-term effects of subcutaneous melatonin implants on intrahypothalamic LHRH content and on pulsatile luteinizing hormone release have been investigated in ganglionectomized male mink. Animals were submitted to bilateral removal of the superior cervical ganglion in mid-April. A preliminary study revealed that plasma LH concentrations remain at a basal level throughout the year following ganglionectomy. In a second experiment, one month after ganglionectomy and transfer from the natural photoperiod environment to short daylengths (LD 4:20), melatonin pellets were subcutaneously implanted to overcome deafferentation of the pineal. Progressive effects of treatment were studied 7 days, 15 days, and one, two and three months after insertion of the melatonin implants. The intra-hypothalamic LHRH content in ganglionectomized mink was at a basal level similar to that observed during seasonally sexual quiescence, or after exposure to inhibitory long days (LD 20:4). A significant and transient elevation in LHRH content was observed already after fifteen days, and also one month after insertion of melatonin implants. This resulted in mean values similar to those observed during the breeding season, or after exposure to stimulatory short days (LD 4:20). A decrease in hypothalamic LHRH content started after two months. No pattern of pulsatile LH secretion was recorded in ganglionectomized untreated mink. A significant increase in all parameters of pulsatile LH secretion was observed fifteen days after the elevation of LHRH content induced by melatonin treatment, and maximum values were reached after two months. Pituitary activity tended to decrease after three months, characterized in particular by a significant decrease in the mean frequency of LH pulses. In addition, the increase in pulsatile characteristics of LH release occurred two months before the peripheral renewal of testicular activity. Apparently, the reproductive endocrine function in ganglionectomized mink treated with melatonin implants is restored more rapidly at the hypothalamic level than at the pituitary or testicular levels.     

Compromised reproductive function in adult female mice selectively expressing mutant ErbB-1 tyrosine kinase receptors in astroglia

The ErbB-1 tyrosine kinase receptor plays critical roles in regulating physiological functions. This receptor-mediated signaling in astroglia has been implicated in controlling female sexual development via activating neurons that release LH-releasing hormone (LHRH), the neuropeptide required for the secretion of LH. It remains unknown whether astroglial ErbB-1 receptors are necessary for maintaining normal adult reproductive function. Here we provide genetic evidence that astroglia-specific and time-controlled disruption of ErbB-1 receptor signaling by expressing mutant ErbB-1 receptors leads to compromised reproduction due to alteration in LHRH neuron-controlled secretion of LH in adult female mice. Therefore, astroglial ErbB-1 receptors are required for controlling LHRH neuronal function and thus maintaining adult reproduction, suggesting that compromised astroglial ErbB-1 signaling may also contribute to reproductive abnormalities in aging females.     

Dynamic alterations in luteinizing hormone-releasing hormone (LHRH) neuronal cell bodies and terminals of adult rats

Summary 1. The decapeptide lueteinizing hormone-releasing hormone (LHRH) is synthesized in neuronal cell bodies diffusely distributed across the basal forebrain and is secreted from neuronal terminals in the median eminence. Once secreted, LHRH enters the portal vessels and is then transported to the anterior pituitary, where it modulates the synthesis and secretion of gonadotropins, which are essential to gonadal function and reproduction.2. Because of the difficulties encountered in studying these diffusely distributed neurons, we have developed strategies which combine immunocytochemistry and computer-assisted techniques to examine individual LHRH neuronal cell bodies, as well as the entire population of LHRH neurons from the diagonal band of Broca to the mammillary bodies. In addition, we have examined LHRH neuronal terminals in the median eminence using computer-assisted imaging techniques to examine individual terminals by electron microscopy or across all rostral-caudal regions of the median eminence by light microscopy. In our most recent studies using confocal microscopy, we have examined the relationships of LHRH terminals to glial processes.3. These studies reveal a very dynamic system of LHRH neuronal cell bodies and terminals. The population of neurons in which LHRH can be detected varies as a function of time after gonadectomy, during the estrous cycle, and during the preovulatory surge of LH during the afternoon of proestrus. Dynamic changes are also observed in LHRH terminals in the median eminence as a function of time after gonadectomy and in specific rostral-caudal regions of the median eminence during the preovulatory surge of LH. Finally, confocal microscopy reveals that LHRH terminals are prevented from contacting the basal lamina of the brain by glial end-feet.4. We are currently examining the hypothesis that these relationships change as a function of endocrine milieu and, therefore, participate in the modulation of LHRH secretion. Ongoing studies focus on defining the sites of action and synergy of multiple sources of regulation of LHRH secretion and their relative importance to ensuring reproductive success.     

The role of nitric oxide in the hypothalamic control of LHRH and oxytocin release, sexual behavior and aging of the LHRH and oxytocin neurons

Nitric oxide (NO) affects reproductive processes both at the level of the brain and reproductive tract and this review is focused on its role as an essential regulator of the hypothalamic control of reproduction. The data gathered indicate that glutamate stimulates noradrenergic neurons which subsequently activate NO-ergic cells via alpha1-adrenergic receptors. The released NO diffuses into luteinizing hormone-releasing hormone (LHRH) terminals where it triggers LHRH secretion by activation of guanylyl cyclase and cyclooxygenase. The NO released by estrogen-stimulated NO-ergic ventromedial neurons plays a crucial role in the regulation of sexual behavior. Furthermore, an increased expression of inducible nitric oxide synthase in the LHRH and oxytocin neurons underlies the destructive action of NO on the aging of the hypothalamic neuroendocrine pathways. Within the hypothalamo-hypophyseal system, NO exerts an inhibitory effect in the control of oxytocin secretion. This action seems to employ an indirect mechanism by which NO may modulate the release of GABA. This review provides an overview of the role of NO in hypothalamic control of LHRH and oxytocin release, aging of the LHRH and oxytocin neurons and sexual behavior.     

Luteinizing hormone response to N6, O2'-dibutyryl adenosine 3',5'-cyclic monophosphate in aged, young and androgenized rats: estradiol effect in vitro

The studies in this report were designed to investigate whether the loss of pituitary luteinizing hormone (LH) responses to N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) in aged noncycling rats was the result of age, endocrine status associated with diestrus, or noncyclic low estrogen status. Pituitary monolayer cultures were prepared from female Fischer 344 rats. Aged (18-month-old) persistent diestrous (PD) rats, young diestrous (D) rats, or noncycling neonatally androgenized-constant estrous (AN-CE) rats were used. Enzymatically dispersed cells were maintained in the same batch of medium supplemented with dextran-coated charcoal adsorbed serums. Total LH contents were 1.75 +/- 0.04, 1.15 +/- 0.03, and 1.71 +/- 0.02 micrograms LH/dish in Day 5 cultures prepared from aged PD, young D, and AN-CE rats, respectively. Incubations with 5 mM DBcAMP for 4 h significantly (P less than 0.05) stimulated LH release in cultures prepared from young D and AN-CE rats but inhibited LH release in cultures prepared from aged PD rats even though a 4-h incubation with 10 nM LH releasing hormone (LHRH) stimulated LH release similarly in cultures of all three types of cells. The loss of DBcAMP-induced LH release in cultures prepared from aged PD rats was reversed by 17 beta-estradiol (E2). This treatment also reduced the basal LH release and increased the cellular LH content. These results indicate that the loss of DBcAMP-induced LH response in the aged rat is not an irreversible aging phenomenon but appears to be associated with the chronically low E2 status of aged PD rats but not young cycling D or noncycling AN-CE rats.     

Influences of age and ovarian follicular reserve on estrous cycle patterns, ovulation, and hormone secretion in the Long-Evans rat

This study examined the influences of aging and reduced ovarian follicular reserve on estrous cyclicity, estradiol (E(2)) production, and gonadotropin secretion. Young virgin and middle-aged (MA) retired breeder female rats were unilaterally ovariectomized (ULO) or sham operated (control). Unilateral ovariectomy of young rats reduced the ovarian follicular reserve by one-half, to a level similar to that found in MA controls. Unilateral ovariectomy of MA females reduced the follicular pool further, to one half of MA controls. The incidence of regular cyclicity was significantly lower in MA ULO females than in young controls, with intermediate cycle frequency in young ULO and MA controls. Among cyclic rats, the magnitude of the proestrous LH surge was highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. Similarly, ovulation rates were highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. While young ULO rats exhibited augmented secondary FSH surges on estrous morning, middle-aged ULO females displayed secondary FSH levels comparable to young controls. The effects of age and reduced follicle number on estrous cyclicity and gonadotropin secretion were not due to altered E(2) secretion, as preovulatory E(2) levels were similar among all groups. Thus, experimental reduction in the follicular reserve exerts acute effects on the preovulatory LH surge, ovulation rate, and estrous cyclicity in both young and MA rats. However, decreased follicle number increases FSH levels only in young rats, indicating aging-related alterations in the feedback regulation of FSH.     

介绍一种LHRH调节LH分泌的离体模型—分散垂体细胞灌流技术

成年雄性 SD 大鼠断头后分离出垂体前叶(anterior pituitary,AP)。用胰蛋白酶消化和机械分散制备 AP 细胞(成活率大于95%)。分散的细胞悬液与生物凝胶混合后装上灌流柱,然后用 M199溶液连续灌流24h 以上。每间隔1~h 给予一次6min 的 LHRH 脉冲式刺激。细胞在此灌流过程中有一个稳定的基础 LH 分泌水平。LHRH 刺激能迅速引起 LH 分泌。对同一剂量 LHRH 的多次刺激可产生相同的 LH 脉冲。在一定的 LHRH 浓度范围内(1×10~(-10)_1×10~(-7)mol/L),LH 分泌与 LHRH 的剂量-效应曲线呈线性。实验结果表明,连续灌流分散的 AP 细胞的技术,优于单层细胞培养和组织块灌流等其他方法,是一种较为理想的研究LHRH 调节 LH 分泌机理的体外模型。