Preovulatory Gonadotropin Surge Research Articles

7934 Tac1-expressing cells in the central premammillary nucleus are essential for induction of the pre-ovulatory LH surge

Abstract Disclosure: S. Leon: None. M. Navarro: None. C. Perdices-Lopez: None. I. Velasco Aguayo: None. E. Torres Jimenez: None. D. Garcia-Galiano: None. V.M. Navarro: None. M. Tena-Sempere: None. Infertility/subfertility is a growing challenge in reproductive medicine. The most common cause of subfertility in females is ovulatory dysfunction. Conditions linked with oligo-/anovulation include polycystic ovary syndrome, hypothalamic amenorrhea and premature ovarian insufficiency. However, the central neuro-endocrine defects affecting the pre-ovulatory LH surge that drives ovulation remain ill defined. While hypothalamic GnRH neurons are the major output pathway for the brain control of ovulation, upstream Kiss1 neurons, particularly in the anteroventral periventricular area (AVPV) of the hypothalamus in rodents, are thought to be crucial for the timed activation of GnRH neurons and generation of the preovulatory surge of gonadotropins. Yet, the major upstream regulators of this Kiss1/GnRH pathway are largely unknown. Substance P (SP, encoded by Tac1), a member of the tachykinin (TAC) family that acts via the receptor, NK1R (encoded by Tacr1), has been shown to centrally regulate gonadotropin release, and, according to our initial data, might modulate the pre-ovulatory surge in mice through its direct action on AVPV Kiss1 neurons. We report herein that Tac1-expressing cells in the ventral premammillary nucleus (PMV) become activated during the preovulatory surge, as denoted by co-expression of c-Fos. Moreover, we document here that these neurons project to key reproductive areas, including the rostral preoptic area (rPOA), AVPV, arcuate nucleus (ARC), and the medial posterodorsal amygdala (MePD). Furthermore, the chemogenetic inhibition of Tac1-expressing neurons in the PMV dramatically decreases the magnitude of the pre-ovulatory LH surge, but has no influence on tonic LH pulsatility. Overall, our findings strongly support a relevant stimulatory role of SP originating from Tac1 neurons at the PMV in the generation of the pre-ovulatory surge of gonadotropins, responsible for ovulation. Presentation: 6/1/2024

Modulatory Effects of Ethinyl Estradiol Plus Drospirenone Contraceptive Pill on Spontaneous and GnRH-Induced LH Secretion

Background: Combined oral contraceptives (COCs) work mostly by preventing the pre-ovulatory gonadotropin surge, but the action of COCs on spontaneous episodic and GnRH (gonadotropin-releasing hormone)-induced LH (luteinizing hormone) release has been poorly evaluated. Oral contraceptives are known to act on the spontaneous hypothalamic–pituitary functions reducing both GnRH and gonadotropin release and blocking ovulation. Aim: To evaluate spontaneous and GnRH-induced LH release during both phases of the menstrual cycle or under the use of the contraceptive pill. Methods: A group of 12 women, subdivided into two groups, volunteered for the study. Group A (n = 6, controls) received no treatments, while Group B (n = 6) received a 21 + 7 combination of ethinyl-estradiol (EE) 30 µg + drospirenone (DRSP) 3 mg. Both groups were evaluated twice: Group A during follicular and luteal phases, Group B during pill assumption and during the suspension interval, performing a pulsatility test, GnRH stimulation test, and hormonal parameters evaluation. Spontaneous and GnRH-induced secretory pulses were evaluated, as well as the instantaneous secretory rate (ISR). Results: COC treatment lowered LH and FSH (follicle stimulating hormone) levels significantly if compared to the follicular phase of spontaneous cycles. During the suspension interval, hormone levels rapidly rose and became comparable to those of the follicular phase of the control group. The LH pulse frequency under COC administration during the suspension interval was similar to that observed during the follicular phase (2.6 ± 0.3 pulses/180 min and 2.3 ± 0.2 pulses/180 min, respectively). The GnRH-induced LH peaks were greater in amplitude and duration than those observed after ISR computation in both groups. The GnRH-induced LH release during the luteal phase of the control subjects was higher than in the follicular phase (51.2 ± 12.3 mIU/mL and 14.9 ± 1.8 mIU/mL, respectively). Conversely, subjects under COC showed a GnRH-induced LH response similar during COC and during the suspension interval. Conclusions: Our data support that the EE + DRSP preparation acts on both spontaneous pulsatile release and GnRH-induced LH release during the withdrawal period of the treatment, and that after 5–7 days from the treatment suspension, steroidal secretion from the ovary is resumed, such as that of androgens. This suggests that in hyperandrogenic patients, a suspension interval as short as 4 days might be clinically better.

The role of Kisspeptin signaling in Oocyte maturation.

Kisspeptins (KPs) secreted from the hypothalamic KP neurons act on KP receptors (KPRs) in gonadotropin (GPN) releasing hormone (GnRH) neurons to produce GnRH. GnRH acts on pituitary gonadotrophs to induce secretion of GPNs, namely follicle stimulating hormone (FSH) and luteinizing hormone (LH), which are essential for ovarian follicle development, oocyte maturation and ovulation. Thus, hypothalamic KPs regulate oocyte maturation indirectly through GPNs. KPs and KPRs are also expressed in the ovarian follicles across species. Recent studies demonstrated that intraovarian KPs also act directly on the KPRs expressed in oocytes to promote oocyte maturation and ovulation. In this review article, we have summarized published reports on the role of hypothalamic and ovarian KP-signaling in oocyte maturation. Gonadal steroid hormones regulate KP secretion from hypothalamic KP neurons, which in turn induces GPN secretion from the hypothalamic-pituitary (HP) axis. On the other hand, GPNs secreted from the HP axis act on the granulosa cells (GCs) and upregulate the expression of ovarian KPs. While KPs are expressed predominantly in the GCs, the KPRs are in the oocytes. Expression of KPs in the ovaries increases with the progression of the estrous cycle and peaks during the preovulatory GPN surge. Intrafollicular KP levels in the ovaries rise with the advancement of developmental stages. Moreover, loss of KPRs in oocytes in mice leads to failure of oocyte maturation and ovulation similar to that of premature ovarian insufficiency (POI). These findings suggest that GC-derived KPs may act on the KPRs in oocytes during their preovulatory maturation. In addition to the intraovarian role of KP-signaling in oocyte maturation, in vivo, a direct role of KP has been identified during in vitro maturation of sheep, porcine, and rat oocytes. KP-stimulation of rat oocytes, in vitro, resulted in Ca2+ release and activation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1 and 2. In vitro treatment of rat or porcine oocytes with KPs upregulated messenger RNA levels of the factors that favor oocyte maturation. In clinical trials, human KP-54 has also been administered successfully to patients undergoing assisted reproductive technologies (ARTs) for increasing oocyte maturation. Exogenous KPs can induce GPN secretion from hypothalamus; however, the possibility of direct KP action on the oocytes cannot be excluded. Understanding the direct in vivo and in vitro roles of KP-signaling in oocyte maturation will help in developing novel KP-based ARTs.

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle.

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography–high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.

Preovulatory follicular fluid and serum metabolome profiles in lactating beef cows with thin, moderate, and obese body condition.

Extremes in body condition reduce fertility and overall productivity in beef cattle herds, due in part to altered systemic metabolic conditions that influence the intrafollicular and uterine environment. Follicular fluid and serum metabolome profiles are influenced by body composition in women and dairy cattle; however, such information is lacking in beef cattle. We hypothesized that body condition score (BCS)-related alterations in the metabolome of preovulatory follicular fluid and serum may influence oocyte maturation while impacting the oviductal or uterine environment. Therefore, we performed a study with the objective to determine the relationship between BCS and the metabolome of follicular fluid and serum in lactating beef cattle. We synchronized the development of a preovulatory follicle in 130 cows of varying BCS. We collected blood and performed transvaginal follicle aspirations to collect follicular fluid from the preovulatory follicle ~18 h after gonadotropin-releasing hormone administration to stimulate the preovulatory gonadotropin surge. We then selected follicular fluid and serum samples from cows with BCS 4 (Thin; n = 14), BCS 6 (Moderate; n = 18), or BCS >8 (Obese; n = 14) for ultra-high performance liquid chromatography-high resolution mass spectrometry. We identified differences in the follicular fluid or serum of thin, moderate, and obese animals based on multiple linear regression. MetaboAnalyst 5.0 was used for enrichment analysis of significant metabolites. We identified 38 metabolites in follicular fluid and 49 metabolites in serum. There were no significant differences in follicular fluid metabolite content among BCS classifications. There were 5, 22, and 1 serum metabolites differentially abundant between thin-obese, moderate-thin, and moderate-obese classifications, respectively (false discovery rate [FDR] < 0.10). These metabolites were enriched in multiple processes including "arginine biosynthesis," "arginine/proline metabolism," and "D-glutamine/D-glutamate metabolism" (FDR < 0.04). Pathways enriched with serum metabolites associated with BCS indicate potentially increased reactive oxygen species (ROS) in serum of thin cows. ROS crossing the blood follicular barrier may negatively impact the oocyte during oocyte maturation and contribute to the reduced pregnancy rates observed in thin beef cows.

Correlation between Pre-Ovulatory Follicle Diameter and Follicular Fluid Metabolome Profiles in Lactating Beef Cows.

Induced ovulation of small pre-ovulatory follicles reduced pregnancy rates, embryo survival, day seven embryo quality, and successful embryo cleavage in beef cows undergoing fixed-time artificial insemination. RNA-sequencing of oocytes and associated cumulus cells collected from pre-ovulatory follicles 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the pre-ovulatory gonadotropin surge suggested reduced capacity for glucose metabolism in cumulus cells of follicles ≤11.7 mm. We hypothesized that the follicular fluid metabolome influences metabolic capacity of the cumulus-oocyte complex and contributes to reduced embryo cleavage and quality grade observed following induced ovulation of small follicles. Therefore, we performed a study to determine the correlation between pre-ovulatory follicle diameter and follicular fluid metabolome profiles in lactating beef cows (Angus, n = 130). We synchronized the development of a pre-ovulatory follicle and collected the follicular contents approximately 20 h after GnRH administration. We then performed ultra-high performance liquid chromatography—high resolution mass spectrometry (UHPLC-HRMS) metabolomic studies on 43 follicular fluid samples and identified 38 metabolites within pre-ovulatory follicles of increasing size. We detected 18 metabolites with a significant, positive correlation to follicle diameter. Individual and pathway enrichment analysis of significantly correlated metabolites suggest that altered glucose and amino acid metabolism likely contribute to reduced developmental competence of oocytes when small pre-ovulatory follicles undergo induced ovulation.

Estrous Cycle Plasticity in the Central Clock Output to Kisspeptin Neurons: Implications for the Preovulatory Surge.

Coordination of ovulation and behavior is critical to reproductive success in many species. During the female estrous cycle, the preovulatory gonadotropin surge occurs when ovarian follicles reach maturity and, in rodents, it begins just before the daily onset of activity, ensuring that ovulation coincides with sex behavior. Timing of the surge relies on projections from the suprachiasmatic nucleus (SCN), the locus of the central circadian clock, to hypothalamic circuits that regulate gonadotropin secretion. The cellular mechanisms through which the SCN controls these circuits and gates the preovulatory surge to the appropriate estrous cycle stage, however, are poorly understood. We investigated in mice the functional impact of SCN arginine-vasopressin (AVP) neuron projections to kisspeptin (Kiss1) neurons in the rostral periventricular area of the third ventricle (RP3VKiss1), responsible for generating the preovulatory surge. Conditional anterograde tracing revealed that SCNAVP neurons innervate approximately half of the RP3VKiss1 neurons. Optogenetic activation of SCNAVP projections in brain slices caused an AVP-mediated stimulation of RP3VKiss1 action potential firing in proestrus, the cycle stage when the surge is generated. This effect was less prominent in diestrus, the preceding cycle stage, and absent in estrus, following ovulation. Remarkably, in estrus, activation of SCNAVP projections resulted in GABA-mediated inhibition of RP3VKiss1 neuron firing, an effect rarely encountered in other cycle stages. Together, these data reveal functional plasticity in SCNAVP neuron output that drives opposing effects on RP3VKiss1 neuron activity across the ovulatory cycle. This might contribute to gating activation of the preovulatory surge to the appropriate estrous cycle stage.

Sequential preovulatory expression of a gonadotropin-releasing hormone-inducible gene, Nr4a3, and its suppressor Anxa5 in the pituitary gland of female rats.

Functional relationship between nuclear receptor subfamily 4 group A member 3 (Nr4a3) and annexin A5 (Anxa5), which are two gonadotropin-releasing hormone (GnRH)-inducible genes, has been established while evaluating pituitary gonadotropes in relation to follicle-stimulating hormone beta (Fshb) expression. However, the physiological variations that arise due to the differential expression of these genes in the pituitary gland during rat estrous cycle remain unknown. This study aimed to evaluate the Nr4a3 and Anxa5 mRNA expression during the estrous cycle in rats in comparison with the expression of the gonadotropin subunit genes, luteinizing hormone beta (Lhb) and Fshb. Nr4a3 mRNA expression showed a single peak at 1400 h of proestrus during the 4-d estrous cycle. Anxa5 mRNA level was elevated along with increased Fshb mRNA expression after the decline of Nr4a3 mRNA until 2300 h. Lhb mRNA expression levels were not significantly changed during the estrous cycle. Notably, addition of a GnRH antagonist at 1100 h completely eradicated luteinizing hormone secretion at 1400 h and 1700 h of proestrus, and significantly reduced the Nr4a3 mRNA expression level at both the time points. These results suggest that GnRH is, at least partly, responsible for the increase in pituitary Nr4a3, and that the interaction between NR4A3 and ANXA5 is required to regulate Fshb expression during the preovulatory gonadotropin surge.

A neural circadian signal essential for ovulation is generated in the suprachiasmatic nucleus during each stage of the oestrous cycle.

What is the central question of this study? Is the suprachiasmatic nucleus the structure that generates the neural circadian signals that occur during every stage of the oestrous cycle, not only pro-oestrus, and are these signals essential for proper regulation of ovulation? What is the main finding and its importance? Transient inhibition of Na+ -dependent action potentials in the suprachiasmatic nucleus by tetrodotoxin microinjection at 14.00h inhibits ovulation irrespective of the stage of the oestrous cycle at which the procedure is performed. Microinjection of saline solution into the suprachiasmatic nucleus has a disruptive effect on ovulation that depends on the stage of the cycle at which it is administered. Reproduction is a highly timed process that depends on both the reproductive and circadian systems. The core oscillator of the latter resides at the suprachiasmatic nuclei (SCN) and it is pivotal for the regulation of the pro-oestrus pre-ovulatory surge of gonadotropins in females. There is evidence to suggest that this system may be involved in the regulation of neuroendocrine events that are essential for ovulation and that occur prior to pro-oestrus. We explored this possibility by transiently inactivating the SCN. Female rats were implanted with guide cannulas aimed at the SCN. After recovery of the oestrous cycle, animals were injected with tetrodotoxin (TTX), artificial cerebrospinal fluid (ACSF) or saline solution while freely moving. Injections were performed at 14.00h of each stage of the oestrous cycle. Animals were killed on the next predicted oestrus day, the number of ova shed was counted and intact rats at oestrus stage were used as absolute control. ACSF did not modify ovulation. Saline solution blocked ovulation in oestrus- and dioestrus-injected rats. Irrespectively of the stage of the oestrous cycle, TTX blocked ovulation. These results lead us to suggest that a neural circadian signal, pivotal for triggering the gonadotropin pre-ovulatory surge, arises from the SCN during the critical window of pro-oestrus. We also suggest that a similar signal, needed for the regulation of other events that are indispensable for proper regulation of ovulation, is also generated in this nucleus during the other stages of the cycle at a similar time.

Clock control of mammalian reproductive cycles: Looking beyond the pre-ovulatory surge of gonadotropins.

Several aspects of the physiology and behavior of organisms are expressed rhythmically with a 24-h periodicity and hence called circadian rhythms. Such rhythms are thought to be an adaptive response that allows to anticipate cyclic events in the environment. In mammals, the circadian system is a hierarchically organized net of endogenous oscillators driven by the hypothalamic suprachiasmatic nucleus (SCN). This system is synchronized by the environment throughout afferent pathways and in turn it organizes the activity of tissues by means of humoral secretions and neuronal projections. It has been shown that reproductive cycles are regulated by the circadian system. In rodents, the lesion of the SCN results on alterations of the estrous cycle, sexual behavior, tonic and phasic secretion of gonadotropin releasing hormone (GnRH)/gonadotropins and in the failure of ovulation. Most of the studies regarding the circadian control of reproduction, in particular of ovulation, have only focused on the participation of the SCN in the triggering of the proestrus surge of gonadotropins. Here we review aspects of the evolution and organization of the circadian system with particular focus on its relationship with the reproductive cycle of laboratory rodents. Experimental evidence of circadian control of neuroendocrine events indispensable for ovulation that occur prior to proestrus are discussed. In order to offer a working model of the circadian regulation of reproduction, its participation on aspects ranging from gamete production, neuroendocrine regulation, sexual behavior, mating coordination, pregnancy and deliver of the product should be assessed experimentally.

Endocrine basis for puberty in heifers and ewes

Maturation processes that culminate in puberty and sexual maturity are initiated before birth, continue through prepuberty (> 50 days before puberty) and peripuberty (50 days before puberty) and are completed early after puberty. The hypothalamus is the primary site of change during transition to sexual maturity. Maturation of the hypothalamus results from decreased negative feedback of oestradiol that leads to increased frequency of release of LH pulses. Increased tonic release of LH pulses during sexual maturation is the primary endocrine factor that regulates the onset of puberty in ewe lambs and heifers. Increased frequency of release of LH pulses enhances development of ovarian follicles which produce enough oestradiol to induce behavioural oestrus and a preovulatory surge of gonadotrophins. In later stages of peripuberty, ovulation or luteinization of follicles results in transient increases in progesterone for shorter periods than is typical for luteal phases of the oestrous cycle of mature ewes and cows. Transient increases in progesterone are not generally preceded by behavioural oestrus. After the demise of the transient luteal structures, puberty is attained with occurrence of the first behavioural oestrus that is accompanied by ovulation and development of a corpus luteum with a typical lifespan. At puberty, all components of the hypothalamic-pituitary-ovarian axis are in place for oestrous cycles to be expressed. Factors that can influence the pubertal rise in release of LH pulses are genotype, gender, season of year when pubertal age is attained, growth or nutritional intake, social cues or treatment with exogenous progestins. Sexual maturation continues after puberty with an enhanced probability of pregnancy occurring from actions of ovarian steroids at the uterus.

Kisspeptin signaling in oocytes is compulsory for ovulation in adult mice

Kisspeptins, encoded by the Kiss1 gene, are crucial brain factors for the control of reproduction, with an essential role in the generation of the pre‐ovulatory surge of gonadotropins and, hence, ovulation. Yet, kisspeptins are also found in peripheral reproductive organs and their receptor, Gpr54, is expressed in various target tissues, including the ovary, where its physiological role remains contentious. Global Gpr54−/− mice are hypogonadotropic and infertile; female null mice display disrupted folliculogenesis and anovulation. However, despite the predominant brain actions of kisspeptins, how much of this phenotype is due to local, and not central, kisspeptin signaling deficiency remains unknown. To address this, we generated a conditional oocyte‐specific Gpr54 deficient mouse line (OoGpr54−/−), by crossing Gdf9‐Cre+ mice with Gpr54loxP/loxP mice. The efficacy of Gdf9‐promoter to drive the expression of Cre in oocytes was verified by a breeding test, and the Gpr54 depletion from oocytes was confirmed with RT‐qPCR. At the time of pubertal transition, OoGpr54−/− females were indistinguishable from their wiltype controls with preserved puberty onset. However, already at 2 months of age, 45% of OoGpr54−/− females showed histological features reminiscent of ovarian failure, with presence of large atretic antral follicles and absence of corpora lutea as an index of anovulation. These features were also associated with disrupted estrous cyclicity characterized by an increase in the length of the estrus phase of the uterine cycle. Penetrance of this phenotype progressed with age, with 55% of OoGpr54−/− females at 3–4 months, 80% at 6 months and 100% at 10 months displaying features of complete premature ovarian insufficiency (POI). In addition, 4‐month‐old OoGpr54−/− female mice displaying POI showed decreased serum estradiol and progesterone levels, yet sustained Gpr54 expression in the hypothalamus and unaltered basal gonadotropin levels. Ovarian RNASeq analyses revealed global changes in expression profiles in OoGpr54−/− mice, including genes involved in cellular communication, steroidogenesis and intracellular signaling pathways. Finally, the anovulatory state of OoGpr54−/− mice was rescued by exogenously administered gonadotropin priming, and was largely prevented by early onset consecutive pregnancies, although multiparous OoGpr54−/− dams showed a slight decrease in fecundity. Our results pinpoint the important role of oocyte kisspeptin signaling in the direct control of ovulation, whose deregulation may also be an underlying cause for POI in women.Support or Funding InformationFiDiPro programme of Academy of Finland and Sigrid Juselius foundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Morphological study of apoptosis in granulosa cells and ovulation in a model of atresia in rat preovulatory follicles.

SummaryPrevious studies have established a model of atresia in preovulatory follicles after stimulation of immature rats with equine chorionic gonadotropin (eCG). This gonadotropin recruits a follicular pool and the deprivation of preovulatory luteinizing hormone (LH) surge induces the atresia in preovulatory follicles. The present study investigated the occurrence of ovulation and provided some morphological features of granulosa cell (GC) apoptosis of atretic follicles at 0, 48, 72 and 120 h after eCG stimulation. Histological sections of ovaries from untreated animals (0 h) showed primordial, primary, secondary and early antral follicles. After 48 h ovaries showed large antral follicles. Preovulatory follicles were observed at 72 h, and two out of five rats displayed cumulus-oocyte complexes (COCs) in the oviducts. All animals exhibited corpora lutea after 120 h. We observed increased estradiol (E2) levels 48 h after eCG treatment that might trigger an endogenous preovulatory gonadotropin surge. Higher progesterone (P4) level, which is the hallmark of a functional corpus luteum, was observed at 120 h. Atresia in secondary and antral follicles was observed by pyknotic granulosa cell nuclei in histology and positive immunolabelling for cleaved caspase 3. We also observed macrophages in secondary and antral follicles in atresia. Transmission electron microscopy revealed GCs with compacted chromatin against the nuclear envelope, nuclear fragmentation, cell shrinkage and fragmentation. No preovulatory follicles showed apoptosis of GCs. In conclusion, our results suggested the occurrence of an endogenous gonadotropin surge, promoting ovulation and preventing atresia of preovulatory follicles.

Temperature gradients in vivo influence maturing male and female gametes in mammals: evidence from the cow.

Since 1980 several reports have indicated that temperatures vary between preovulatory follicles and other ovarian tissues in rabbit, cow, pig and human. However, these observations did not achieve prominence; they were regarded as artefacts due to the use of anaesthetics and open surgery (laparotomy). Recently, without resorting to anaesthesia or surgery, direct measurements of temperature in preovulatory follicles have been performed in the cow by means of a thermistor probe introduced into the antrum under ultrasonic guidance. Such follicles revealed a mean antral (follicular fluid) temperature 0.74°C and 1.54°C cooler than uterine surface and rectal temperatures respectively in ovulating cows, whereas no such temperature differences were detected in non-ovulating cows. Cows are predominantly monovular and preovulatory follicles attain a diameter of 15-22mm or more. These features and the timescale of response to the preovulatory gonadotrophin surge make them a valuable model for the human preovulatory follicle. Temperature gradients are interpreted primarily in a context of final maturation of gametes immediately before the onset of fertilisation. Preovulatory follicular temperature in women could be assessed by a comparable approach and might become a valuable selection guide for oocyte viability.

Oocyte maturation and quality: role of cyclic nucleotides.

The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP - inhibiting the hydrolysis of intra-oocyte cAMP - and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect - the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options.

Ghrelin suppresses the GnRH-induced preovulatory gonadotropin surge in dairy heifers

Ghrelin, a known growth hormone (GH) secretagogue, alters gonadotropin secretion in many species. Our objectives were to study the effects of ghrelin, on GH, LH, FSH secretion, and on luteal function of the ensuing estrous cycle in cattle. The estrous cycles of eight heifers were synchronized with progesteron releasing intravaginal device, and ovulation was induced with GnRH. Eight animals were treated with 1.5 μg kg−1 bovine ghrelin (group Ghr, n = 4) or saline (group C, n = 4). Starting with the first ghrelin injection, 13 blood samples were collected over a 4-hour period for the determination of ghrelin, GH, LH, and FSH concentration. Progesterone levels were measured in samples collected every other day after estrus expression. Data were analyzed by repeated measures of ANOVA followed by Bonferroni post hoc testing and t test. In group Ghr, ghrelin concentration increased significantly 15 minutes after the first injection and remained in elevated levels until the 90th minute after the last injection. At the time of third ghrelin injection, GH was significantly higher in the Ghr group compared with C (17.1 ± 1.3 vs. 2.6 ± 0.3 ng mL−1, P < 0.0001). Similar differences were found for the next three samples collected 15, 30, and 60 minutes later; no difference was evident after 90 minutes. In group Ghr, the area under the curve for LH and FSH were significantly reduced compared with the ones of group C (266 ± 10.3 vs. 331.9 ± 7.3, P = 0.007 and 102.3 ± 2.0 vs. 134.9 ± 5.5, P < 0.005 for LH and FSH respectively). At particular time points the concentration of the two gonadotrophins in group Ghr was significantly lower than those of group C (15, 30, 45, 75, and 90 and 60, 75, 90, 120, and 150 minutes after GnRH administration for LH and FSH respectively). The duration of the following estrous cycle was shorter (P = 0.004) in group Ghr (19.0 ± 0.4 days) compared with C (21.8 ± 0.5 days). In days 4, 6, 8, 10, and 14, progesterone concentration was lower (P < 0.05) in group Ghr compared with C; similarly the progesterone area under the curve for group Ghr (113.1 ± 4.8) was suppressed (P = 0.007) compared with that of C (141 ± 4.8). These results imply that ghrelin acts on pituitary causing impaired response to the GnRH stimulus, and it is likely to affect luteinization of the cellular compartment of the preovulatory follicle, and/or to suppress steroidogenetic activity of the luteal cells.

Variaciones hormonales del ciclo menstrual normal

Se revisan las variaciones hormonales que caracterizan el ciclo menstrual normal, señalándose que depende de una adecuada interrelación hipotálamo-hipófiso-ovárica. Se da cuenta de los cambios diarios de las gonadotrofinas, estrógenos y progesterona, medidos por análisis radioinmunológico en la sangre, obtenida a lo largo de 7 ciclos menstruales normales. Se pudo constatar que las concentraciones de FSH y LH son ligeramente mayores en la fase folicular que en la luteal, exhibiendo todos ellas una elevación mesocíclica coincidente en la fase ovulatoria tanto de LH como de FSH. El estradiol mostró una elevación importante previa al pico ovulatorio de gonadotrofinas y otra en la fase luteal. La progesterona se mantuvo en una concentración baja durante la fase folicular incrementándose notablemente desde la aparición del pico ovulatorio de gonadotrofinas. Se discute el significado de estos cambios hormonales.

Loss of Ntrk2/Kiss1r signaling in oocytes causes premature ovarian failure.

Neurotrophins (NTs), once believed to be neural-specific trophic factors, are now known to also provide developmental cues to non-neural cells. In the ovary, NTs contribute to both the formation and development of follicles. Here we show that oocyte-specific deletion of the Ntrk2 gene that encodes the NTRK2 receptor (NTRK2) for neurotrophin-4/5 and brain-derived neurotrophic factor (BDNF) results in post-pubertal oocyte death, loss of follicular organization, and early adulthood infertility. Oocytes lacking NTRK2 do not respond to gonadotropins with activation of phosphatidylinositol 3-kinase (PI3K)-AKT-mediated signaling. Before puberty, oocytes only express a truncated NTRK2 form (NTRK2.T1), but at puberty full-length (NTRK2.FL) receptors are rapidly induced by the preovulatory gonadotropin surge. A cell line expressing both NTRK2.T1 and the kisspeptin receptor (KISS1R) responds to BDNF stimulation with activation of Ntrk2 expression only if kisspeptin is present. This suggests that BDNF and kisspeptin that are produced by granulosa cells (GCs) of periovulatory follicles act in concert to mediate the effect of gonadotropins on Ntrk2 expression in oocytes. In keeping with this finding, the oocytes of NTRK2-intact mice fail to respond to gonadotropins with increased Ntrk2 expression in the absence of KISS1R. Our results demonstrate that the preovulatory gonadotropin surge promotes oocyte survival at the onset of reproductive cyclicity by inducing oocyte expression of NTRK2.FL receptors that set in motion an AKT-mediated survival pathway. They also suggest that gonadotropins activate NTRK2.FL expression via a dual communication pathway involving BDNF and kisspeptin produced in GCs and their respective receptors NTRK2.T1 and KISS1R expressed in oocytes.

Human ghrelin decreases pituitary response to GnRH in superovulated ewes

In addition to its metabolic role, ghrelin has been found to suppress luteinizing hormone secretion in many species acting mainly at the hypothalamic level. The objectives of the present study were to test the hypothesis that besides its effects on the hypothalamic level, ghrelin exerts a direct action on the pituitary. Twelve cycling ewes were synchronized, using progestagen intravaginal sponges and superovulated using eCG. At the time of sponge withdrawal, animals were allocated into two groups, ghrelin-treated (Gh) and control. Two days after the sponge removal, GnRH was given to synchronize ovulations. Simultaneously with GnRH treatment, animals of the Gh group received the first of four treatments of acylated human ghrelin at a dose of 6 μg/kg body weight iv; three additional treatments of ghrelin iv were given every 15 minutes thereafter. Control animals received saline iv. Blood samples were collected before challenge (−30 and 0 minutes) and at 30, 60, 75, 90, 105, 120, 135, 150, and 180 minutes after GnRH treatment, and were analyzed for LH, FSH, estradiol, progesterone, insulin, and insulin-like growth factor-I concentrations. Ghrelin treatment attenuated GnRH-induced a preovulatory surge of both gonadotrophins, with the effect being greater for LH. No difference was detected for insulin, estradiol, and progesterone concentrations, and insulin-like growth factor-I levels were increased in the Gh group. Our results imply that in sheep, ghrelin conducts specific regulatory effects on the GnRH/LH axis, and provide for the first time strong evidence that besides its central action, ghrelin might regulate gonadotrophin release acting at the pituitary level.

The activity and copy number of mitochondrial DNA in ovine oocytes throughout oogenesis in vivo and during oocyte maturation in vitro

Mitochondria are responsible for the production of ATP, which drives cellular metabolic and biosynthetic processes. This is the first study to quantify the mtDNA copy number across all stages of oogenesis in a large monovulatory species, it includes assessment of the activity of mitochondria in germinal vesicle (GV) and metaphase II (MII) oocytes through JC1 staining. Primordial to early antral follicles (n = 249) were isolated from the sheep ovarian cortex following digestion at 37°C for 1 h and all oocytes were disaggregated from their somatic cells. Germinal vesicle oocytes (n = 133) were aspirated from 3- to 5-mm diameter antral follicles, and mature MII oocytes (n = 71) were generated following in vitro maturation (IVM). The mtDNA copy number in each oocyte was quantified using real-time PCR and showed a progressive, but variable increase in the amount of mtDNA in oocytes from primordial follicles (605 ± 205, n = 8) to mature MII oocytes (744 633 ± 115 799, n = 13; P < 0.05). Mitochondrial activity (P > 0.05) was not altered during meiotic progression from GV to MII during IVM. The observed increase in the mtDNA copy number across oogenesis reflects the changing ATP demands needed to orchestrate cytoskeletal and cytoplasmic reorganization during oocyte growth and maturation and the need to fuel the resumption of meiosis in mature oocytes following the pre-ovulatory gonadotrophin surge.