Reproductive Arrest Research Articles

Gene expression during Drosophila melanogaster egg development before and after reproductive diapause

BackgroundDespite the importance of egg development to the female life cycle in Drosophila, global patterns of gene expression have not been examined in detail, primarily due to the difficulty in isolating synchronised developmental stages in sufficient quantities for gene expression profiling. Entry into vitellogenesis is a key stage of oogenesis and by forcing females into reproductive diapause we are able to arrest oogenesis at the pre-vitellogenic stages. Releasing females from diapause allows collection of relatively synchronous developing egg populations and an investigation of some of the transcriptional dynamics apparent before and after reproductive diapause.ResultsFocusing on gender-biased transcription, we identified mechanisms of egg development suppressed during reproductive dormancy as well as other molecular changes unique to the diapausing female. A microarray based analysis generated a set of 3565 transcripts with at least 2-fold greater expression in females as compared to control males, 1392 such changes were biased during reproductive dormancy. In addition, we also detect 1922 up-regulated transcriptional changes after entry into vitellogenesis, which were classified into discrete blocks of co-expression. We discuss some of the regulatory aspects apparent after re-initiation of egg development, exploring the underlying functions, maternal contribution and evolutionary conservation of co-expression patterns involved in egg production.ConclusionAlthough much of the work we present is descriptive, fundamental aspects of egg development and gender-biased transcription can be derived from our time-series experiment. We believe that our dataset will facilitate further exploration of the developmental and evolutionary characteristics of oogenesis as well as the nature of reproductive arrest in Drosophila.

Genetic basis for reproductive diapause is correlated with life history traits within the Culex pipiens complex

The evolution of late season reproductive arrest (diapause) among female Culex pipiens mosquitoes allows them to overwinter in temperate climates, while females of the sibling species Culex quinquefasciatus do not exhibit the diapause phenotype. We present results for quantitative trait loci (QTL) analyses of two independent segregating populations derived from crosses between C. pipiens and C. quinquefasciatus. QTL for diapause and three life history traits were identified and compared for genome positions and gene effects. Using a combination of composite interval mapping, single-marker analysis and all possible subsets regression, we identified multiple QTL for each trait, totalling 14 and 17 QTL for each population, respectively. Individual QTL across traits often mapped to similar genome locations, suggesting these traits may be controlled in part by genes with pleiotropic effects or multiple tightly linked genes. The majority of QTL were intermediate in magnitude in that they explained 10-25% of the phenotypic variation. The majority of QTL showed overdominance effects. We suggest that this could impact natural populations, as increased heterosis across hybrid zones may allow populations to adapt to environmental conditions via stabilizing selection, and yet maintain species identity outside these regions because of strong morphological integration, wherein related traits evolve as an integrated unit.

Natural variation in Drosophila melanogaster diapause due to the insulin-regulated PI3-kinase

This study links natural variation in a Drosophila melanogaster overwintering strategy, diapause, to the insulin-regulated phosphatidylinositol 3-kinase (PI3-kinase) gene, Dp110. Variation in diapause, a reproductive arrest, was associated with Dp110 by using Dp110 deletions and genomic rescue fragments in transgenic flies. Deletions of Dp110 increased the proportion of individuals in diapause, whereas expression of Dp110 in the nervous system, but not including the visual system, decreased it. The roles of phosphatidylinositol 3-kinase for both diapause in D. melanogaster and dauer formation in Caenorhabditis elegans suggest a conserved role for this kinase in both reproductive and developmental arrests in response to environmental stresses.

Endocrine regulation of the reproductive arrest in the long-winged females of a flightless bug, Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae)

Mechanisms of the suppression of gonadotropic activity of the corpus allatum (CA) in females were com- pared with those previously reported for either or non-diapause females by reciprocal transplanta- tions of the neuroendocrine complexes (comprising the brain-suboesophageal ganglion-corpora cardiaca-CA). The denervated CA stimulated reproduction in most females of all experimental groups suggesting an inhibition of the CA via nervous connections with the brain. The inhibition of the CA within the transplanted neuroendocrine complex was measured by the reproductive performance of feeding recipient females deprived of their own CA. The complex from non-diapause females stimulated reproduction in 58.3-78.9% of recipients suggesting that the inhibition of the CA was mostly overcome by the stimulating internal milieu of feeding females. In contrast, the macropterous complex stimulated reproduction in only 18.8-37.5% of recipients, similar to the diapause brachypterous complex (32.0%). The results indicate that the macropterism, similar to the diapause, is associated with a considerably lower responsiveness of the neuroendocrine complex to humoral stimulation by feeding compared to the responsivenes of the starving complex from non-diapause females. On the other hand, the CA of females is of intermediate size between that of the feeding non-diapause and females, similar to the CA of the non-diapause females deprived of food. Overall, the data suggest that the suppression of the CA activity results from a combination of the diapause-like refractoriness of the neuroendocrine complex with the starvation-like inhibition of the CA growth. Regulation of the CA activity is discussed in relation to the oogenesis-flight syndrome recorded for flying wing- polymorphic species of insects.

Detecting Genes Expressed at Different Stages of Reproductive Arrest in Brassica oleracea

Cauliflower (Brassica oleracea var. italica) and Broccoli (Brassica oleracea var. botrytis) differ mainly in the stage of reproductive arrest. Cauliflower curd is an inflorescence meristem, while broccoli arrests just before anthesis. Arabidopsis studies led to the hypothesis that a mutant BoCAL allele arrested cauliflower earlier. Later, a mutant in BoAP1 was found to have similar effects. These partially redundant genes, and several identified since, are present in multiple copies in B. oleracea. Understanding their role in the arrest requires quantification of transcript abundance analysis by real-time PCR. Designing selective PCR primers is a critical first step in the process. Designs were based on alignment among the genes of interest (MADS-box genes BoCAL, BoAP1, FUL, and the non MADS-box genes LFY and TFL1) and their paralogs. The high sequence similarity (some over 95%) makes the target transcripts difficult to distinguish. Therefore, primers were designed mostly for targets in the 3'UTR region in order to gain specificity. Short amplicons, 68bp to 200bp, were required for the high PCR efficiency required to quantify these low-abundance transcripts. Primers were evaluated by conventional RT-PCR and real-time PCR. By altering temperature, Total RNA was isolated from plants that were arrested at three developmental stages, inflorescence meristem (cauliflower), floral meristem (intermediate), and floral bud (broccoli) by varying temperature. RT-PCR products were single bands of the expected size, despite the high homology between genes under study. Real-time melting curve analysis (fluorescence derivative vs. melting temperature) corroborated the presence of a single amplicon. The identity of products was confirmed by sequencing and restriction enzyme digestion.

RNA interference-mediated knockdown of a GATA factor reveals a link to anautogeny in the mosquito Aedes aegypti.

Blood feeding tightly regulates the reproductive cycle in anautogenous mosquitoes. Vitellogenesis (the synthesis of yolk protein precursors) is a key event in the mosquito reproductive cycle and is activated in response to a blood meal. Before blood feeding, Aedes aegypti is in a state of reproductive arrest during which the yolk protein precursor genes (YPPs) are repressed. The regulatory region of the major YPP gene vitellogenin (Vg) has multiple GATA-binding sites required for the high expression level of this gene. However, a GATA factor (AaGATAr) likely acts as a repressor, preventing activation of this gene before a blood meal. Here we report in vivo data confirming the role of AaGATAr as a repressor of the Vg gene at the state of previtellogenic arrest. Using an RNA interference (RNAi)-mediated technique in conjunction with the Sindbis viral expression system, we show that knockdown of the AaGATAr gene results in an increased basal level of expression of the Vg gene and an elevated response to the steroid hormone 20-hydroxyecdysone in mosquitoes in a state of arrest. These experiments have revealed a component in the molecular mechanism by which anautogeny is maintained in A. aegypti.

Period gene expression in relation to seasonality and circadian rhythms in the linden bug, Pyrrhocoris apterus (Heteroptera)

Wild females of Pyrrhocoris apterus exhibit seasonal changes in neuroendocrine activity and, consequently, reproduction. Long days (18 h light/6 h dark) (LD) stimulate reproduction, whereas short days (12 h light/12 h dark) (SD) induce reproductive arrest (diapause). This study reveals how photoperiod influences the expression of the circadian clock gene, period (per) in the insect's head. There is only a weak diurnal rhythm in per mRNA expression under LD and SD. However, levels of per mRNA are consistently higher (up to 10-fold) under SD than under LD. The influence of photoperiod on per gene expression is linked to a developmental output (diapause vs. reproduction); mutant females, reproducing under both LD and SD, show low per mRNA levels under both photoperiodic conditions. Thus, the magnitude of per gene expression may be important to the translation of photoperiodic signals into a hormonal message. Levels of per mRNA are related to properties of locomotor activity rhythms. Low per mRNA levels (displayed by wild females in LD and mutant females in both LD and SD) are associated with long free-running periods (τ~26-27 h) and late peaks of activity (ψR,L~10-12 h), whereas high per mRNA levels coincide with short free-running periods (τ~24 h) and early peaks of activity (ψR,L~4-6 h). Overall, the data provide a background for a molecular approach to the long-standing question about the role of the circadian system in insect photoperiodism.

Eyestalk ablation-induced androgenic gland activity and gonad development in the freshwater prawns Macrobrachium nipponense (De Haan, 1849)

Summary Effects of eyestalk ablation on androgenic gland (AG) protein synthesis and the following gonad development were determined in reproductive and non-reproductive adult males of the freshwater prawn Macrobrachium nipponense. During the non-reproductive season, the AG cells from intact prawns were characterized by a small amount of vacuolated cytoplasm and highly pycnotic nuclei. The AG cells from unilateral eyestalk ablated prawns (UEA) consisted of developing rough surfaced endoplasmic reticulum (rER) a molting cycle after the operation. The AG cells of bilateral eyestalk ablated prawns (BEA) showed a more extensively developed rER and Golgi complex, which are similar in appearance to AG cells from normal reproductive prawns. The gonads from reproductive prawns were in active spermatogenesis. Eyestalk ablation prompted the progress, particularly in the BEA. The gonads from non-reproductive prawns were under reproductive arrest. The eyestalk ablation induced the non-reproductive prawns to be reproductive. The reproductive performance induced by BEA was as successful as in the normal reproductive prawns in terms of gonado-somatic index (GSI). The GSI of the phased changed prawn was 1.21 whereas the GSI of normal prawns during the reproductive season was 1.10. These data taken together with the AG cell changes suggest that eyestalk ablations appear to induce precocious male gonad development by stimulating AG cells from the prawn.

Possible involvement of ecdysteroids in photoperiodically induced suppresion of ovarian development in a Japanese strain of the migratory locust, Locusta migratoria

In a Japanese population of Locusta migratoria, adult females become reproductively inactive under crowding and long days (LD) and reproductively active under crowding and short days (SD). The identity and titre of ecdysteroids in the haemolymph and ovaries from adult females reared under SD and LD were investigated by RIA/HPLC. The effects of exogenous juvenile hormone (JH) III treatments on the termination of such reproductive arrest and ecdysteroid contents in LD females were also examined. In general, ecdysteroid titres in both haemolymph and ovaries were significantly higher in reproductively active SD females than in reproductively inactive LD females. A clear difference was also observed in oocyte growth between SD and LD individuals. JH III applications (three consecutive topical applications, 150 μg per insect per day from day 3) stimulated ovarian development in LD females and significantly increased the haemolymph and ovarian ecdysteroids to a level comparable to that of reproductively active SD adult females.

Diel changes in adipokinetic response and walking activity of Pyrrhocoris apterus (Heteroptera) in relation to physiological status and wing dimorphism

Three experimental groups of adult females of Pyrrhocoris apterus (reproductive brachypterous, diapausing brachypterous, and macropterous with non-diapause type of reproductive arrest) were analysed for their dose dependency of adipokinetic response, sensitivity of the fat body to Pya-AKH and locomotor activity during a 24 hour cycle. The greatest adipokinetic response (lipid elevation after hormonal treatment) was observed in macropterous, lower in diapausing brachypterous and the lowest in reproductive brachypterous females. Macropterous females also exhibited the highest activity of walking, while the lowest locomotor activity was found in diapausing brachypters. Diel changes of adipokinetic response were recorded in all three experimental groups, with maximal response occurring during the day at 6-7 h after the onset of light. On the contrary, the minimal adipokinetic response was found in the night and early after the onset of light. No significant differences in diel rhythms were observed between groups exposed to various day lengths. Diel changes in locomotor activity were found only in reproductive brachypterous females and females of macropterous morph, and showed a diurnal rhythm with only one peak of activity during a 24 hour cycle. In the latter, the rhythmic pattern of walking activity was positively correlated with diel changes of adipokinetic response. This indicates an important role of Pya-AKH in dispersal strategy of macropterous females of this heteropteran.

Physiological effects of methyl farnesoate and pyriproxyfen on wintering female crayfish Cherax quadricarinatus

Methyl farnesoate (MF), the predominant juvenile hormone-like compound of crustaceans, was found in the hemolymph of female Cherax quadricarinatus crayfish. Administration of MF to C. quadricarinatus females during their winter reproductive arrest period had no effect on reproduction; however, it did have a tendency to accelerate molting. However, since MF caused increased mortality (∼47% survival in the high MF treatment), we were not able to draw definitive conclusions regarding its physiological affect. In contrast, administration of pyriproxyfen, a juvenile hormone analog, did not cause significant mortality (95% survival in the high pyriproxyfen treatment), although it accumulated in high quantities in the hepatopancreas and, to a lesser extent, muscle tissue, ovaries and gills. The highest dose of pyriproxyfen used in this study, 20 μg/gram animal body weight/week, caused a delay in spawning, which became statistically significant from the seventeenth week. This dose of pyriproxyfen caused a tendency of acceleration of molting without any effect on molt increment. The results of this study show that pyriproxyfen does not seem to be toxic to the crayfish, even in relatively high doses, and might affect the energetic balance between molt and reproduction.

Locomotor activity in adult Pyrrhocoris apterus (Heteroptera) in relation to sex, physiological status and wing dimorphism

Summary The pattern of locomotor (walking) activity was studied in adult males and females of short-winged (brachypterous) and long-winged (macropterous) morph of the flightless bug Pyrrhocoris apterus (Linnaeus) (Heteroptera: Pyrrhocoridae) under constant laboratory conditions. Walking activity was measured with a computerized video system and analysed with respect to sex, physiological status (reproduction, diapause and reproductive arrest of non-diapause type) and wing dimorphism of the bugs. The largest duration was observed in the macropterous females with reproductive arrest of non-diapause type (average 6 h per day) and the shortest duration in diapausing brachypterous females and males (average less than 2 h per day). This was reflected also in the overall time spent by walking during the first 14 days after imaginal ecdysis. The time spent by walking significantly increased in the macropterous morph as the bugs aged, whereas in diapausing brachypters the time spent by walking decreased with age. No linear relationship between walking activity and age was found in reproductive brachypterous morph. The bugs of all experimental groups moved mostly during the photophase and were almost inactive during the scotophase. Thus, walking activity in P. apterus is diurnal, irrespective of the wing morph, physiological status, sex and age. Contrary to the macropterous morph, where the locomotor activity of females during photophase was significantly higher than in males, no significant differences were found between the locomotor activities of brachypterous males and females. The observed differences in locomotor activity are discussed in relation to different roles of two wing morphs in the life history of this heteropteran.

High-density lipoprotein associated with secondary vitellogenesis in the hemolymph of the crayfish Cherax quadricarinatus

The high-density lipoproteins LPI and LPII were isolated from the hemolymph of the crayfish Cherax quadricarinatus by gradient ultracentrifugation and high-performance liquid chromatography (HPLC). Both lipoproteins contained a carotenoid moiety. LPI is comprised of a single polypeptide with an approximate molecular mass of 96 kDa. LPII was composed of two similar native components, LPIIa and LPIIb, both having polypeptides of 80 and 177 kDa. Both under natural conditions and after endocrine manipulations, LPI was present in males and in females, regardless of the female reproductive stage. LPII was present only in secondary-vitellogenic females, but not during the winter reproductive arrest period. LPII was also absent from young females that had received androgenic gland implants. LPII also appeared in the hemolymph of intersex individuals from which the androgenic gland had been removed. It is therefore suggested that LPII serves as a marker indicating the onset of secondary vitellogenesis in C. quadricarinatus females.

Differences in adipokinetic response of Pyrrhocoris apterus (Heteroptera) in relation to wing dimorphism and diapause

SummaryThree experimental groups of adult females (reproductive and diapausing brachypters, and macropters with reproductive arrest) of Pyrrhocoris apterus (Linnaeus) (Heteroptera: Pyrrhocoridae) from a temperate population were analysed for their adipokinetic responses. The adipokinetic response, expressed as an increase of haemolymph lipids after injection of adipokinetic hormone from Locusta migratoria (Lom‐AKH‐I), was assessed in relation to age, wing dimorphism and type of reproductive arrest. Two pmols of Lom‐AKH‐I were used for determination of adipokinetic responses. The increase of haemolymph lipids in all experimental groups of females induced by this dose of the hormone was comparable with that induced by crude extract of the bug’s own corpora cardiaca. The level of adipokinetic response after injection of 2 pmol of Lom‐AKH‐I was significantly higher in macropterous and diapausing brachypterous females than in reproductive brachypterous females. However, significantly higher contents of haemolymph lipids in control macropterous females than those found in the control reproductive and diapausing brachypterous females of the corresponding age revealed wing‐morph‐related differences in lipid metabolism. The observed wing morph‐ and diapause‐related differences in the content of haemolymph lipids and adipokinetic response, respectively, are discussed in relation to the different roles of two wing morphs in the life history of this heteropteran.

Wing morph-related physiological differences in adults of temperate population of Pyrrhocoris apterus (L.) (Heteroptera: Pyrrhocoridae)

The reproductive and diapausing adult females of brachypterous morph and macropterous females with reproductive arrest of non-diapause type, originating from the laboratory cultures of Pyrrhocoris apterus, were studied for their feeding and drinking behaviour, digestive enzyme activities, and carbohydrate and lipid contents. The highest feeding and drinking activities were observed in reproductive brachypters, the lowest in macropters. Macropters also differed from brachypters by lower activities of gut lipase, peptidase and protease, lower concentration of haemolymph sugars, and lower weight of fat body, which probably reflects their low feeding activity. The total content of fat body lipids was also lower in macropters (0.6 mg) than in reproductive and diapausing brachypters (4.6 and 7.5 mg, respectively) on day 14. A very high amount of glycogen was found in the fat body of diapausing brachypters, 363 μg on day 14, as opposed to 15 and 80 μg in macropterous and reproductive brachypterous females, respectively. The obtained data indicate that the most important difference between macropterous and brachypterous females with different types of reproductive arrest consists of an enhanced mobilization of lipids for dispersal in macropters and accumulation of energetic reserves for hibernation in brachypters.

Feeding behaviour, digestive physiology and lipid content in macropterous females of Pyrrhocoris apterus (L.) (Heteroptera: Pyrrhocoridae)

Abstract. Macropterous females of Pyrrhocoris apterus (L.) reared under short‐day conditions (LD 12:12 h) were analysed for temporal patterns of feeding and drinking behaviour, activities of digestive enzymes in the gut, and lipid and glycogen content in the haemolymph and fat body. Peaks of drinking activity were recorded at days 3, 7 and 10 during the first 14 days after imaginal ecdysis. Feeding activity peaked on the third day, ceasing completely after the fourth day of adult life. Esterase, protease, amylase and aminopeptidase activities exhibited the highest overall activity in the first days after imaginal emergence; then enzyme activities decreased. In the fat body, the content of lipids was highest on day 5, then a decrease of about 40% was observed at day 14; the amount of glycogen was highest on day 1 at 11 μg of glucose equivalents/mg of fat body, then decreased to 2 μg at day 14 after the imaginal moult. In the haemolymph, the lipid content rose until day 8 when it reached almost 0.3 μmol/μl; at day 14 the value was slightly lower. The association of fasting with reproductive arrest in macropterous females of P. apterus, accompanied by a decrease in digestive enzyme activities and a mobilization of lipid reserves from the fat body, was demonstrated.

Targeting gene therapy vectors to CNS malignancies.

Gene therapy offers significant advantages to the field of oncology with the addition of specifically and uniquely engineered mechanisms of halting malignant proliferation through cytotoxicity or reproductive arrest. To confer a true benefit to the therapeutic ratio (the relative toxicity to tumor compared to normal tissue) a vector or the transgene it carries must selectively affect or access tumor cells. Beyond the selective toxicities of many transgene products, which frequently parallel that of contemporary chemotherapeutic agents, lies the potential utility of targeting the vector. This review presents an overview of current and potential methods for designing vectors targeted to CNS malignancies through selective delivery, cell entry, transport or transcriptional regulation. The topic of delivery encompasses physical and pharmaceutic means of increasing the relative exposure of tumors to vector. Cell entry based methodologies are founded on increasing relative uptake of vector through the chemical or recombinant addition of ligand and antibody domains which selectively bind receptors expressed on target cells. Targeted transport involves the potential for using cells to selectively carry vectors or transgenes into tumors. Finally, promoter and enhancer systems are discussed which have potential for selectivity activating transcription to produce targeted transgene expression or vector propagation.

Dormancy in the stalk-eyed fly, Diopsis longicornis Macq., a pest of rice in sub-Saharan Africa

Dormancy is described for adults of the stalk-eyed fly, Diopsis longicornis Macquart (Diopsidae: Diptera) in the tropical savanna of coastal Guinee, West Africa. Populations of diopsids maintained themselves within each chosen refugium throughout the dry season unless the site dried up. Sex ratio within the populations at each refugium remained constant throughout the dry period. Slightly more females than males were found (56%), and of these only 0.8% were gravid. Movement of stalk-eyed fly adults from the rice field to the refugia corresponded with the end of the rice cropping period (stage of ripening and harvest) at the end of the wet season (late October to November), whereas movement from the refugia to the rice field occurred towards the end of the dry season Gate (May/early June), but well in advance of the rice crop planting (July and August) of the following wet season. Insects retained their state of reproductive arrest in the dry season even when offered suitable oviposition sites, i.e. rice plants. Secondary host plants were investigated as possible oviposition sites and food sources for adult flies.

Variations in the Diapause Characteristics of Amblyseius womersleyi Schicha(Acari: Phytoseiidae).

The incidence of diapause at 15-20°C/9L15D was determined in 6 populations of the phytoseiid mite, Amblyseius womersleyi Schicha, collected from south-central and southern Japan. Diapause in phytoseiid mites is characterized by reproductive arrest in females, however, some females induced by short daylength and low temperature exhibited incomplete diapause as follows: 1)oviposition ceased after they laid a few eggs (“ switching” into diapause) ; and 2) the pre-oviposition period was much longer than that of females reared under a long-day photoperiod (“ reproductive delay” ) . In the populations from south-central Honshu and northern Kyushu, temperature greatly affected the diapause expression: almost 100% of the individuals entered diapause at 15° C and the percentages of diapause decreased with increasing temperature to 45-75% at 18°C and 10-40% at 20°C On the other hand, in the populations from Okinawa the percentages of diapause remained low (0-25%) over the temperature range tested, and temperature only slightly affected the expression of diapause. In particular, the percentage observed in the Tokashiki population was only 10.3%, even at 15° C. These results show that the diapause in A. womersleyi is a quantitative threshold trait and that variation in the diapause capacity exists within populations, as well as among populations from different localities. In some phytoseiid species, the importance of a thermoperiod for diapause induction has been shown, but in A. womersleyi a thermoperiod in combination with a short daylength (scotophase coincided with cool phase) held no stimulation for diapause induction.

Role of the Thyroid Gland in Seasonal Reproduction: Thyroidectomy Blocks Seasonal Suppression of Reproductive Neuroendocrine Activity in Ewes*

Seasonal reproductive transitions in ewes are generated endogenously and are synchronized by annual changes in photoperiod. Previous evidence indicates that thyroidectomy prevents the transition to anestrus in ewes maintained in a fixed day length, suggesting that the thyroid is needed for endogenously generated reproductive arrest. Here we tested the hypothesis that the thyroid is required for endogenous seasonal suppression of the neuroendocrine mechanism that regulates pulsatile LH secretion. Ewes were thyroidectomized (n = 6) in summer, 6 weeks before the onset of the breeding season, or they remained thyroid intact (n = 6). They were housed in a simulated natural photoperiod until the winter solstice; thereafter, they remained on that photoperiod (10 h of light, 14 h of darkness). To monitor pulsatile LH secretion, the ewes were ovariectomized and implanted with estradiol, and LH was measured in both frequent (every 6 min for 4 h) and infrequent (twice weekly) blood samples. In this model, high LH indicates low response to estradiol negative feedback and reproductive induction; low LH signifies high response to estradiol negative feedback and reproductive arrest. LH levels (samples twice weekly) rose some 50-fold in both groups concurrently at the start of the breeding season in September. Frequent sampling in midbreeding season (autumn) revealed that both thyroidectomized and thyroid-intact ewes exhibited frequent LH pulses, with no group difference, in either the presence or absence of the estradiol implant. A marked group difference, however, emerged at the end of the breeding season. LH fell to basal values in thyroid-intact ewes (onset of low values Feb 3 +/- 8 days), whereas levels remained elevated in thyroidectomized ewes through the end of the study (April 26). At this time, thyroidectomized ewes had more frequent LH pulses than thyroid-intact ewes both in the presence and absence of estradiol. The circadian pattern of melatonin secretion and the seasonal change in PRL were found to be unaffected by thyroidectomy. These observations support the hypothesis that the thyroid is necessary for endogenous suppression of neuroendocrine mechanisms that generate LH pulses, a suppression crucial for the transition to anestrus.