Fetal programming: prenatal androgen disrupts positive feedback actions of estradiol but does not affect timing of puberty in female sheep

We studied the impact of prenatal androgen exposure on the timing of onset of puberty, maintenance of cyclicity in the first breeding season, and the LH surge mechanism in female sheep. Pregnant sheep were injected with testosterone propionate (100 mg i.m.) twice each week from Day 30 to Day 90 (D30-90) or from Day 60 to Day 90 (D60-90) of gestation (term = 147 days). Concentrations of plasma progesterone and gonadotropins were measured in blood samples collected twice each week from control (n = 10), D60-90 (n = 13), and D30-90 (n = 3) animals. Rate of weight gain and initiation of estrous behavior were also monitored. After the first breeding season, when the animals entered anestrus, competency of the gonadotropin surge system to respond to estradiol positive feedback was tested in the absence or presence of progesterone priming for 12 days. Prenatally androgenized females had similar body weight gain and achieved puberty (start of first progestogenic cycle) at the same time as controls. Duration of the breeding season and the number of cycles that occurred during the first breeding season were similar between control and prenatally androgenized sheep. In contrast, prenatal exposure to androgens compromised the positive feedback effects of estradiol. Onset of LH/FSH surges following the estradiol stimulus was delayed in both groups of androgenized ewes compared with the controls in both the absence and presence of progesterone priming. In addition, the magnitude of LH and FSH surges in the two animals that surged in the D30-90 group were only one third and one half, respectively, of the magnitudes observed in the control and D60-90 groups. The present findings indicate that disruption of the surge system can account for the fertility problems that occur during adulthood in prenatally androgenized sheep.     

Sexual behaviour and LH secretion in spayed androgenized ewes after a s?ngle injection of testosterone or oestradiol-17beta

The behavioural and endocrine responses to single injections of 50 or 500 microgram oestradiol-17beta or 5 mg testosterone were recorded in spayed (control) ewes and in spayed ewes exposed to testosterone between Days 30 and 80 or Days 50 and 100 of prenatal life, The control ewes showed oestrus after injections on 17/18 occasions. The androgenized ewes showed poorer oestrous responses to each hormone although rams showed interest in the ewes. Masculine sexual and aggressive behaviour was shown by the androgenized ewes given either steroid. Both steroids caused a reduction in the plasma LH levels of all the ewes (negative feedback), followed by a preovulatory-type surge (positive feedback). The peak LH values were significantly lower (P is less than 0.05) in the Day 50-100 androgenized ewes than in the controls. It is concluded that prenatal androgenization causes a qualitative shift in the sexual behaviour of ewes from the female type to the male type and affects the sensitivity of the brain to "positive feedback" by steroids.     

Resumption of gonadotrophin release during the post-partum period in suckling and non-suckling ewes

The post-partum secretion of LH, FSH and prolactin was monitored in 15 suckling and 6 non-suckling Préalpes du Sud ewes lambing during the breeding season by measuring plasma hormone concentrations daily at 6-h intervals and also weekly at 20-min intervals for 6 h from parturition to resumption of regular cyclic ovarian activity. There was a constant phenomenon in the resumption of normal patterns of FSH and LH secretion: there was a rise in FSH values culminating on average on Day 4 post partum and returning subsequently to values observed during the oestrous cycle, and concurrently an increase in the frequency and amplitude of LH pulses more progressive in suckling than in non-suckling ewes which led to an elevation of LH mean concentrations and occurrence of an LH surge. Since neither the FSH secretory pattern nor FSH mean values differed between suckling and non-suckling ewes, the results suggested that LH pulsatile pattern was a major limiting factor for the resumption of normal oestrous cycles. Before regular oestrous cycles resumed other changes in preovulatory LH surges also occurred: (i) they increased in duration and probably in amplitude; (ii) they were preceded by an acceleration in LH pulse frequency and a large decrease in FSH values as in normal cyclic ewes; and (iii) at least in non-suckling ewes they occurred concurrently with a prolactin surge.     

Plasma follicle stimulating hormone in Merino ewes immunized with an inhibin-enriched fraction from bovine follicular fluid

Plasma FSH concentrations were measured in Merino ewes immunized with either an inhibin-enriched preparation from bovine follicular fluid (bFFI) or bovine serum albumin. When compared during the normal oestrous cycle, ewes reimmunized three times with bFFI and which showed increased ovulation rates before the experiment had significantly elevated plasma FSH concentrations on Day 13–14 and at Day 2 of the subsequent cycle. There was a positive correlation (P < 0.05) between plasma FSH concentration and the ovulation rate of the ewes in previous cycles (during the period of immunization) and in the cycle under investigation. In a larger group of ewes immunized against bFFI, which showed a variable increase in ovulation rate, there was no comparable increase in plasma FSH concentration when compared with control ewes in the follicular phase of the cycle.By contrast, when luteolysis was induced by a prostaglandin analogue the bFFI-immunized ewes had lower plasma FSH concentrations than control ewes immediately before and after the preovulatory LH surge. This decrease was significant in the period 9–21 h after the LH surge (P < 0.05–0.01) so that the onset of the second FSH peak was delayed.When the ewes were ovariectomized, the post-castration rise in plasma FSH concentration (but not LH) was delayed for a period of 24 h in bFFI-immunized ewes relative to controls.These experiments show that immunization of ewes with an inhibin-like fraction of bFF does not lead to consistently elevated plasma FSH. However, such ewes have altered feedback regulation leading to differential responses of FSH to prostaglandin-induced luteolysis and to castration.     

Differential estradiol requirement for the induction of estrus behavior and the luteinizing hormone surge in two breeds of sheep

For a better understanding of the mechanisms that lead to the preovulatory GnRH/LH surge and estrus behavior, the minimum estradiol (E) requirements (dose and duration) to induce each of these events were determined and compared between two breeds of ewes having either single (Ile de France) or multiple (Romanov) ovulations. The ewes were initially studied during a natural estrus cycle, and were then ovariectomized and run through successive artificial estrus cycles. For these artificial cycles the duration and amplitude of the follucular phase E increase were manipulated by E implants. Under all conditions, the onset of estrus behavior was similar in the two breeds, although its duration was longer in Romanov ewes. While a moderate E signal (6 cm for 12 h) induced an LH surge in 10/10 Ile de France ewes, a larger E signal (12 cm for 12 h) was minimally effective in Romanov ewes (4/10). Additional studies revealed that a small E signal (3 cm for 6 h) induced full estrus behavior in all Romanov ewes but was completely ineffective in Ile de France animals (0/10). Higher doses and mostly longer durations of the E signal (12 cm for 24 h) were required to induce a surge in all the Romanov ewes. These results demonstrate a clear difference in the E requirement for the induction of estrus behavior and the LH surge between breeds of ewes that have different ovulation rates. These data provide compelling evidence that, in one breed, the neuronal systems that regulate both events require different estrogen signals.     

Gonadotropin secretion during prolonged hyperprolactinemia: basal secretion and the stimulatory feedback effect of estrogen

Inoculation of cyclic female rats with the prolactin (Prl)/growth hormone-secreting pituitary tumor, MtT.W15, resulted in a cessation of estrous cyclicity within 5--10 days. Associated with this acyclicity was a persistently low serum concentration of estradiol and marked increases in both circulating Prl and progesterone. At Day 26 of acyclicity, basal serum luteinizing hormone (LH) values measured in samples taken every 20 min from 0900--1100 h were significantly reduced when compared to cyclic, nontumor animals on diestrus Day 2. There was no difference in basal follicle-stimulating hormone (FSH) concentrations. In a separate group of acyclic, tumor-bearing females 42--56 days after transplantation, a single s.c. injection of 20 micrograms estradiol benzoate (EB) at 1030 h elicited significant increases in both serum LH and FSH values between 1700 and 1830 h on the next day. The magnitude of the LH surge was reduced and that of FSH was increased in tumor-bearing animals when compared to cyclic, nontumor females given a similar EB injection on diestrus Day 1. These results demonstrate that chronic hyperprolactinemia is associated with inhibition of basal LH secretion and ovarian estrogen production and an increase in circulating progesterone concentrations. Nevertheless, the stimulatory feedback effects of estrogen on LH and FSH release are still present and functioning in acyclic female rats under chronically hyperprolactinemic conditions. These data suggest that the cessation of regular ovulatory cycles associated with hyperprolactinemia may be due to a deficiency of LH and/or estrogen secretion, but not to a lack of central nervous system response to the stimulatory feedback action of estrogen.     

Fetal programming: testosterone exposure of the female sheep during midgestation disrupts the dynamics of its adult gonadotropin secretion during the periovulatory period

Prenatal exposure of the female sheep to excess testosterone (T) leads to hypergonadotropism, multifollicular ovaries, and progressive loss of reproductive cycles. We have determined that prenatal T treatment delays the latency of the estradiol (E2)-induced LH surge. To extend this finding into a natural physiological context, the present study was conducted to determine if the malprogrammed surge mechanism alters the reproductive cycle. Specifically, we wished to determine if prenatal T treatment 1) delays the onset of the preovulatory gonadotropin surge during the natural follicular phase rise in E2, 2) alters pulsatile LH secretion and the dynamics of the secondary FSH surge, and 3) compromises the ensuing luteal function. Females prenatally T-treated from Day 60 to Day 90 of gestation (147 days is term) and control females were studied when they were approximately 2.5 yr of age. Reproductive cycles of control and prenatally T-treated females were synchronized with PGF2alpha, and peripheral blood samples were collected every 2 h for 120 h to characterize cyclic changes in E2, LH, and FSH and then daily for 14 days to monitor changes in luteal progesterone. To assess LH pulse patterns, blood samples were also collected frequently (each 5 min for 6 h) during the follicular and luteal phases of the cycle. The results revealed that, in prenatally T-treated females, 1) the preovulatory increase in E2 was normal; 2) the latencies between the preovulatory increase in E2 and the peaks of the primary LH and FSH surges were longer, but the magnitudes similar; 3) follicular-phase LH pulse frequency was increased; 4) the interval between the primary and secondary FSH surges was reduced but there was a tendency for an increase in duration of the secondary FSH surge; but 5) luteal progesterone patterns were in general unaltered. Thus, exposure of the female to excess T before birth produces perturbances and maltiming in periovulatory gonadotropin secretory dynamics, but these do not produce apparent defects in cycle regularity or luteal function. To reveal the pathologies that lead to the eventual subfertility arising from excess T exposure during midgestation, studies at older ages must be conducted to assess if there is progressive disruption of neuroendocrine and ovarian function.     

Induction of ovulation in the acyclic postpartum ewe following continuous, low-dose subcutaneous infusion of GnRH

Pituitary and ovarian responses to subcutaneous infusion of GnRH were investigated in acyclic, lactating Mule ewes during the breeding season. Thirty postpartum ewes were split into 3 equal groups; Group G received GnRH (250 ng/h) for 96 h; Group P + G was primed with progestagen for 10 d then received GnRH (250 ng/h) for 96 h; and Group P received progestagen priming and saline vehicle only. The infusions were delivered via osmotic minipumps inserted 26.6 +/- 0.45 d post partum (Day 0 of the study). Blood samples were collected for LH analysis every 15 min from 12 h before until 8 h after minipump insertion, then every 2 h for a further 112 h. Daily blood samples were collected for progesterone analysis on Days 1 to 10 following minipump insertion, then every third day for a further 25 d. In addition, the reproductive tract was examined by laparoscopy on Day -5 and Day +7 and estrous behavior was monitored between Day -4 and Day +7. Progestagen priming suppressed (P < 0.05) plasma LH levels (0.27 +/- 0.03 vs 0.46 +/- 0.06 ng/ml) during the preinfusion period, but the GnRH-induced LH release was similar for Group G and Group P + G. The LH surge began significantly (P < 0.05) earlier (32.0 +/- 3.0 vs 56.3 +/- 4.1 h) and was of greater magnitude (32.15 +/- 3.56 vs 18.84 +/- 4.13 ng/ml) in the unprimed than the primed ewes. None of the ewes infused with saline produced a preovulatory LH surge. The GnRH infusion induced ovulation in 10/10 unprimed and 7/9 progestagen-primed ewes, with no significant difference in ovulation rate (1.78 +/- 0.15 and 1.33 +/- 0.21, respectively). Ovulation was followed by normal luteal function in 4/10 Group-G ewes, while the remaining 6 ewes had short luteal phases. In contrast, each of the 7 Group-P + G ewes that ovulated secreted progesterone for at least 10 d, although elevated plasma progesterone levels were maintained in 3/7 unmated ewes for >35 d. Throughout the study only 2 ewes (both from Group P + G) displayed estrus. These data demonstrate that although a low dose, continuous infusion of GnRH can increase tonic LH concentrations sufficient to promote a preovulatory LH surge and induce ovulation, behavioral estrus and normal luteal function do not consistently follow ovulation in the progestagen-primed, postpartum ewe.     

Regulation of LH beta subunit mRNA in the sheep pituitary gland during different feedback states of estradiol

The feedback effects of gonadal steroids on the amounts of in vitro translated luteinizing hormone (LH) beta subunit were examined using cell-free assays. These amounts were then correlated with serum and pituitary concentrations during various feedback states. RNA was prepared, translated and products identified by immunoprecipitation and gel electrophoresis. The amounts of beta subunit varied in a pattern similar to that observed for alpha subunit. In ovariectomized ewes, the amounts of beta were 2–3X those seen in negative feedback groups and slightly more than those seen in animals exhibiting an LH surge. The pituitary LH concentration in ovariectomized ewes was also higher than those seen in the other groups, however, the serum concentrations in the positive feedback group were the highest of all groups. These results provide evidence for: 1) a separate, but coordinate, control of gonadotropin subunit synthesis; and 2) a contribution of subunit synthesis to the effects of positive and negative steroid feedback on pituitary LH amounts.     

Progesterone blockade of the positive feedback effects of 17β oestradiol in the ewe

Ovariectomized ewes (n = 24) were treated with implants that resulted in circulating concentrations of progesterone and 17β-oestradiol similar to those seen in intact ewes in the luteal phase of an oestrous cycle. Progesterone implants were left in for 10 days, and 17β-oestradiol implants for 14 days. Twelve of these ewes received daily injections of 17β-oestradiol in oil (i.m.) at doses sufficient to cause a surge release of luteinizing hormone (LH) in the absence of progesterone. The other 12 ewes were treated daily with vehicle (oil). Following progesterone withdrawal on Day 10, each group of 12 ewes was divided into three subgroups. Ewes in each subgroup of the groups treated daily with 17β-oestradiol or vehicle, received an injection of either 17β-oestradiol (oil i.m.), gonadotrophin-releasing hormone (GnRH) (saline, i.v.) or vehicle, 24 h after progesterone withdrawal. Following progesterone withdrawal, no LH surge was detected in ewes treated with vehicle. Surge secretion of LH was detected in ewes of all other groups. The data suggested that in progesterone-treated ewes, daily exposure to stimulatory doses of 17β-oestradiol did not desensitize the hypothalamic pituitary axis to the positive feedback effects of 17β-oestradiol. Daily exposure to 17β-oestradiol did not suppress pituitary responsiveness to GnRH. It was concluded that circulating concentrations of progesterone, similar to those seen during the luteal phase of an oestrous cycle in intact ewes, may prevent all necessary components of the LH surge secretory mechanism from responding to 17β-oestradiol.     

Changes in pulsatile LH secretion and the positive and negative feedback actions of oestradiol after administration of a GnRH agonist in the ovariectomized ewe

Administration of a GnRH agonist (5 micrograms) every 12 h to long-term ovariectomized ewes for 5 or 10 days during the breeding season suppressed mean LH levels from around 6 to 1 ng/ml on Days 1 and 4 after treatment; on Day 1 after treatment LH pulse frequency and amplitude were lower than pretreatment values. On Day 4 after treatment LH pulse frequency was restored to pretreatment levels (1 per h) whereas LH pulse amplitude had only slightly increased from 0.5 to 1 ng/ml, a value 25% of that before treatment. This increase in amplitude was greater the shorter the duration of treatment. Ovariectomized ewes treated with the agonist for 5 days exhibited both negative and positive feedback actions after implantation of a capsule containing oestradiol; however, compared to control ewes treated with oestradiol only, the positive and negative feedback actions of oestradiol were blunted. These results suggest that the recovery of tonic LH concentrations after GnRH agonist-induced suppression is limited primarily by changes in LH pulse amplitude. The results also demonstrate that the feedback actions of oestradiol are attenuated, but not blocked, by GnRH agonist treatment.     

The sexual behaviour of prenatally androgenized ewes observed in the field

Pregnant ewes were implanted with 1 g testosterone between Days 30-80, 50-100, 70-120 or 90-140 of gestation. Treatments which began on Days 30, 50 or 70 resulted in the birth of androgenized females which failed to show regular oestrous cycles in adult life, but which exhibited patterns of male-like behaviour. This was most marked in the Day 50-100 and 70-120 groups, whereas complete masculinization of the external genitalia was confined to the Day 30-80 group. Animals in the Day 90-140 group had regular oestrous cycles although they showed slight enhancement of masculine behaviour compared to the control ewes. These results demonstrate that androgenization involves both a suppression of female behavioural patterns, and the development of male patterns; these are not mutually exclusive.     

Behavioral and endocrine responses of hair sheep ewes exposed to different mating stimuli around estrus

Hair sheep ewes were used to evaluate the influence of various levels of mating stimuli on the duration and timing of estrus and LH concentrations around estrus. Ewes were treated with PGF2alpha (15 mg, im) 10 d apart. At the time of the second PGF2alpha treatment (Day 0) ewes were placed in groups and exposed to different types of mating stimuli. One group of ewes (n = 16) was exposed to an epididymectomized ram (RAM), a second group of ewes (n = 16) was exposed to an epididymectomized ram wearing an apron to prevent intromission (APRON) and a third group of ewes (n = 17) was exposed to an androgenized ovariectomized ewe (T-EWE). Jugular blood samples were collected from ewes at 6-h intervals through Day 5. Plasma was harvested and LH concentration was determined by RIA. The ewes were observed at 6-h intervals to detect estrus. A ewe was considered to be out of estrus when she no longer stood to be mounted by the teaser animal. There was no difference (P > 0.10) in the proportion of ewes expressing estrus (79.6%) or having an LH surge (85.7%) among the treatments. Neither the time to estrus nor the duration of estrus were different (P > 0.10) among APRON, RAM or T-EWE groups (41.6+/-3.8 vs 43.6+/-3.6 vs 46.1+/-3.6 h, respectively, and 26.5+/-2.2 vs 24.8+/-2.3 vs 30.5+/-2.2 h, respectively). The time to LH surge was similar (P > 0.10) among APRON, RAM and T-EWE groups (51.2+/-4.5 vs 51.2+/-4.7 vs 52.7+/-4.5 h, respectively). The magnitude of the LH surge was similar (P > 0.10) in the T-EWE, APRON and RAM ewes (99.7+/-4.9 vs 87.2+/-4.9 vs 85.8+/-5.0 ng/mL, respectively). The time from estrus to the LH surge was not different (P > 0.10) among APRON, RAM or T-EWE ewes (10.1+/-2.2 vs 9.8+/-2.3 vs 11.6+/-2.3 h, respectively). These results show that the expression and duration of estrus are not influenced by different types of mating stimuli in hair sheep ewes. In addition, the timing and the magnitude of LH release does not appear to be influenced by mating stimuli around the time of estrus.     

Reproductive physiology and development of artificial insemination technology in killer whales (Orcinus orca)

Research was conducted to define the basic reproductive physiology of killer whales (Orcinus orca) and to use this knowledge to facilitate the development of artificial insemination procedures. The specific objectives were 1) to determine the excretory dynamics of urinary LH and ovarian steroid metabolites during the estrous cycle; 2) to evaluate the effect of an exogenously administered, synthetic progesterone analog on reproductive hormone excretion; 3) to validate the use of transabdominal ultrasound for ovarian evaluation and timing of ovulation; 4) to examine the quality of semen after liquid storage and cryopreservation; and 5) to develop an intrauterine insemination technique. Based on urinary endocrine monitoring of 41 follicular phases and 26 complete cycles from five females, estrous cycles were 41 days long and comprised a 17-day follicular phase and a 21-day luteal phase. A consistent temporal relationship was observed between peak estrogen conjugates and the LH surge, the latter of which occurred approximately 0.5 days later. Two animals placed on oral altrenogest (three separate occasions for 30, 17, and 31 days, respectively) excreted peak urinary estrogen concentrations 25 days after withdrawal that were followed by sustained elevations in urinary pregnanediol-3alpha-glucuronide excretion. Mean preovulatory follicle diameter was 3.9 cm (n = 6), and ovulation occurred 38 h (n = 5) after the peak of the LH surge. Based on visual estimates of motility, liquid-stored semen maintained 92% of its raw ejaculate sperm motility index (total progressive motility x kinetic rating [0-5 scale, where 0 = no movement and 5 = rapid progressive movement]) when held at 4 degrees C for 3 days postcollection. Semen cryopreserved using a medium freezing rate demonstrated good postthaw total motility (50%), progressive motility (94%), and kinetic rating (3.5). Insemination during eight estrous cycles resulted in three pregnancies (38%), two from liquid-stored and one from cryopreserved semen. Two calves were delivered after gestation lengths of 552 and 554 days, respectively. These data demonstrate the potential of noninvasive endocrine monitoring combined with serial ultrasonography to improve our understanding of the reproductive biology of cetaceans. This fundamental knowledge was essential for ensuring the first successful conceptions, resulting in live offspring, using artificial insemination in any cetacean species.     

Human aldosterone synthase: recombinant expression in E. coli and purification enables a detailed biochemical analysis of the protein on the molecular level

Recent experiments from our laboratory are consistent with the idea that hypothalamic astrocytes are critical components of the central nervous system (CNS) mediated estrogen positive feedback mechanism. The "astrocrine hypothesis" maintains that ovarian estradiol rapidly increases free cytoplasmic calcium concentrations ([Ca(2+)](i)) that facilitate progesterone synthesis in astrocytes. This hypothalamic neuroprogesterone along with the elevated estrogen from the ovaries allows for the surge release of gonadotropin-releasing hormone (GnRH) that triggers the pituitary luteinizing hormone (LH) surge. A narrow range of estradiol stimulated progesterone production supports an "off-on-off" mechanism regulating the transition from estrogen negative feedback to estrogen positive feedback, and back again. The rapidity of the [Ca(2+)](i) response and progesterone synthesis support a non-genomic, membrane-initiated signaling mechanism. In hypothalamic astrocytes, membrane-associated estrogen receptors (mERs) signal through transactivation of the metabotropic glutamate receptor type 1a (mGluR1a), implying that astrocytic function is influenced by surrounding glutamatergic nerve terminals. Although other putative mERs, such as mERβ, STX-activated mER-Gα(q), and G protein-coupled receptor 30 (GPR30), are present and participate in membrane-mediated signaling, their influence in reproduction is still obscure since female reproduction be it estrogen positive feedback or lordosis behavior requires mERα. The astrocrine hypothesis is also consistent with the well-known sexual dimorphism of estrogen positive feedback. In rodents, only post-pubertal females exhibit this positive feedback. Hypothalamic astrocytes cultured from females, but not males, responded to estradiol by increasing progesterone synthesis. Estrogen autoregulates its own signaling by regulating levels of mERα in the plasma membrane of female astrocytes. In male astrocytes, the estradiol-induced increase in mERα was attenuated, suggesting that membrane-initiated estradiol signaling (MIES) would also be blunted. Indeed, estradiol induced [Ca(2+)](i) release in male astrocytes, but not to levels required to stimulate progesterone synthesis. Investigation of this sexual differentiation was performed using hypothalamic astrocytes from post-pubertal four core genotype (FCG) mice. In this model, genetic sex is uncoupled from gonadal sex. We demonstrated that animals that developed testes (XYM and XXM) lacked estrogen positive feedback, strongly suggesting that the sexual differentiation of progesterone synthesis is driven by the sex steroid environment during early development. This article is part of a Special Issue entitled 'Neurosteroids'.     

Differential changes in the mechanisms controlling luteinizing hormone and prolactin secretion in middle-aged female rats

Serum luteinizing hormone (LH) and prolactin (PRL) concentrations were measured in young (3-4 month old) and middle-aged (10-12 month old) intact female rats on proestrus, in ovariectomized rats after two estrogen injections (estradiol benzoate; EB, 10 micrograms/100 g body weight, s.c.) or after preoptic stimulation in EB-primed ovariectomized rats. Only animals showing regular 4-day estrous cycles were selected for the experiment. The magnitude of proestrous LH surge was significantly smaller in middle-aged than in young rats. Two BE injections, at noon on Days 0 and 3, in ovariectomized middle-aged rats failed to induce surges in LH secretion on Day 4 whereas the same treatment produced LH surges in ovariectomized young rats. The preoptic electrochemical stimulation (50 microA for 60 sec) produced a prompt rise in serum LH levels in ovariectomized EB-primed young but not in middle aged rats. The preoptic stimulation with a larger current (200 microA) induced LH secretin in middle-aged rats. In none of these situations serum PRL concentrations were different between young and middle-age rats. These results suggest differential aging rates in the preoptic mechanisms governing LH and PRL secretion in the rat. The function of the preoptic ovulatory center in responding to the estrogen positive feedback action and inducing LH secretion may become impaired and independent of the PRL control mechanism, even before the regular estrous cycle terminates.     

Circannual cycles of luteinizing hormone and prolactin secretion in ewes during prolonged exposure to a fixed photoperiod: evidence for an endogenous reproductive rhythm

Circulating patterns of luteinizing hormone (LH) and prolactin (PRL) were monitored for 5 yr in ewes maintained either outdoors in natural conditions or indoors in a fixed, short photoperiod (8L:16D). The ewes were ovariectomized and each was treated with a Silastic implant containing estradiol to provide a fixed negative feedback signal to the reproductive neuroendocrine axis. Serum concentrations of LH and PRL were subjected to a statistical algorithm developed for the purpose of detecting hormone cycles. In ewes maintained outdoors, serum concentrations of both hormones underwent high amplitude cycles with a period no different from 365 days. Among ewes maintained in the fixed photoperiod, unambiguous cycles of LH and PRL persisted through the 5 yr of exposure to short days. Period of these cycles differed from 365 days. Further, the LH cycles became desynchronized among ewes housed together and desynchronized with respect to the LH cycles in ewes kept outdoors. These findings document the existence of an endogenous circannual rhythm of reproductive neuroendocrine function in ewes.     

Fluctuations in the plasma levels of the follicle-stimulating hormone during estrous cycle, anestrus, gestation and lactation in the ewe: evidence for an endogenous rhythm of FSH release

Two experiments were carried out to analyse FSH secretion in the ewe. The first was a long-term study during which four ewes under controlled photoperiods were checked for plasma concentrations of FSH twice daily for a period of 16 months. They were successively anestrous, cycling, gestating and lactating. The results suggested that an endogenous secretion rhythm of FSH persisted throughout each of the physiological states of the ewes. The periodic cycles of FSH production lasted about 5 days during anestrus and gestation but extended to about 6 days during estrus. One of the three waves of secretion we noted during one cycle was represented by the two periovulatory surges, the first coincident with the LH peak, the second occuring 30-40 h later. Plasma levels of FSH were similar during estrous cycles and anestrus, whereas the FSH secretion decreased gradually throughoug gestation. During lactation, large differences were observed among animals before the recovery of cyclic ovarian activity. The second experiment consisted of frequent blood sampling (every ten minutes) of eight ewes for 6 hours during anestrus. FSH was secreted differently compared to LH. No pulsatile production of FSH was demonstrated and no increase in FSH levels was seen at the time of the episodic LH surge.     

Role of short days in timing the onset and duration of reproductive activity in ewes under artificial photoperiods

The objective of this work was to evaluate the role of short photoperiod in timing the onset and duration of reproductive activity in ewes. The perception of photoperiod was disrupted by pinealectomy following transfer from long (17L:7D) to short (8.5L:15.5D) photoperiod and the subsequent reproductive response was monitored. Ovariectomized ewes given Silastic implants containing estradiol-17 beta were exposed to long days until Day 0 (May 24) and then were allocated to the following groups (n = 5-6/group): Group 1) short-day control--moved to short days; Groups 2 to 5) pinealectomy after 0, 30, 60, or 90 short days, respectively; Group 6) long-day hold--kept on long days; Group 7) long days after 60 short days--moved to short days on Day 0 and returned to long days on Day 60. Six ewes kept outdoors served as additional controls. Reproductive neuroendocrine activity was assessed from plasma LH concentrations, high values being indicative of the breeding season and low values indicative of anestrus. Time of reproductive neuroendocrine activity onset (LH rise) did not differ among animals in the 7 groups kept indoors, but was advanced (p less than 0.05) relative to that of ewes outdoors. In contrast, duration of the LH elevation differed among ewes in groups kept indoors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of third ventricular injection of beta-endorphin on luteinizing hormone surges in female rat: sites and mechanisms of opioid actions in the brain

The effects of third ventricular injection of beta-endorphin (beta-EP) on spontaneous, brain stimulation-induced and estrogen-induced LH surges were studied in the adult female rat. It was found that beta-EP blocked the preovulatory surge of LH release and ovulation, while it did not affect LH release in response to LH-RH injection. The site of the beta-EP blockade of ovulation was proved to be in the brain. Beta-EP completely blocked ovulatory LH release induced by the electrochemical stimulation of the medial amygdaloid nucleus and medial septum-diagonal band of Broca, but failed to block ovulation due to the stimulation of the medial preoptic area (MPO) or median eminence, though serum LH levels after the MPO stimulation were inhibited by beta-EP. In the spayed rats treated with estradiol benzoate (EB) on Day 1 and 4 of experiment, beta-EP given on Day 5 blocked the LH surge that normally occurred on that day and led to a compensatory surge of LH on the following day. Moreover, the LH surge on Day 5 was inhibited by beta-EP given either on Day 1 or Day 4. Present data suggest that beta-EP may act in inhibiting the preovulatory LH surges not only by suppressing the preoptic-tuberal LH-RH activities but also by affecting the initiation and development of stimulatory feedback of estrogen in the central nervous system.