Chrysanthi Fergani

Effects of stress on reproduction in ewes

Stressors, such as poor body condition, adverse temperatures or even common management procedures (e.g., transport or shearing) suppress normal oestrus behaviour and reduce ewe fertility. All these events are co-ordinated by endocrine interactions, which are disrupted in stressful situations. This disruption is usually temporary in adult ewes, so that, when prevailing conditions improve, normal fertility would resume. Imposition of an experimental stressor (shearing, transport, isolation from other sheep, injection of endotoxin or insulin or cortisol infusion) suppresses GnRH/LH pulse frequency and amplitude. Part of the cause is at the pituitary, but effects on GnRH/LH pulse frequency and the GnRH/LH surge are mediated via the hypothalamus. It is not yet clear whether delays in the surge are caused by interruption of the oestradiol signal-reading phase, the signal transmission phase or GnRH surge release. Stressors also delay the onset of behaviour, sometimes distancing this from the onset of the pre-ovulatory LH surge. This could have deleterious consequences for fertility.

Insulin: Its role in the central control of reproduction☆

Insulin has long been recognized as a key regulator of energy homeostasis via its actions at the level of the brain, but in addition, plays a role in regulating neural control of reproduction. In this review, we consider and compare evidence from animal models demonstrating a role for insulin for physiological control of reproduction by effects on GnRH/LH secretion. We also review the role that insulin plays in prenatal programming of adult reproduction, and consider specific candidate neurons in the adult hypothalamus by which insulin may act to regulate reproductive function. Finally, we review clinical evidence of the role that insulin may play in adult human fertility and reproductive disorders. Overall, while insulin appears to have a significant impact on reproductive neuroendocrine function, there are many unanswered questions regarding its precise sites and mechanisms of action, and their impact on developing and adult reproductive neuroendocrine function.

Kisspeptin, c-Fos and CRFR type 2 expression in the preoptic area and mediobasal hypothalamus during the follicular phase of intact ewes, and alteration after LPS.

Increasing estradiol concentrations during the late follicular phase stimulate sexual behavior and the GnRH/LH surge, and it is known that kisspeptin signaling is essential for the latter. Administration of LPS can block these events, but the mechanism involved is unclear. We examined brain tissue from intact ewes to determine: i) which regions are activated with respect to sexual behavior, the LH surge and LPS administration, ii) the location and activation pattern of kisspeptin cells in control and LPS treated animals, and iii) whether CRFR type 2 is involved in such disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and control animals were killed at 0 h, 16 h, 31 h or 40 h (n=4-6/group) after progesterone withdrawal (time zero). At 28 h, other animals received endotoxin (LPS; 100 ng/kg) and were subsequently killed at 31 h or 40 h (n=5/group). LH surges only occurred in control ewes, during which there was a marked increase in c-Fos expression within the ventromedial nucleus (VMN), arcuate nucleus (ARC), and medial preoptic area (mPOA), as well as an increase in the percentage of kisspeptin cells co-expressing c-Fos in the ARC and mPOA compared to animals sacrificed at all other times. Expression of c-Fos also increased in the bed nucleus of the stria terminalis (BNST) in animals just before the expected onset of sexual behavior. However, LPS treatment increased c-Fos expression within the VMN, ARC, mPOA and diagonal band of broca (dBb), along with CRFR type 2 immunoreactivity in the lower part of the ARC and median eminence (ME), compared to controls. Furthermore, the percentage of kisspeptin cells co-expressing c-Fos was lower in the ARC and mPOA. Thus, we hypothesize that in intact ewes, the BNST is involved in the initiation of sexual behavior while the VMN, ARC, and mPOA as well as kisspeptin cells located in the latter two areas are involved in estradiol positive feedback only during the LH surge. By contrast, disruption of sexual behavior and the LH surge after LPS involves cells located in the VMN, ARC, mPOA and dBb, as well as cells containing CRFR type 2 in the lower part of the ARC and ME, and is accompanied by inhibition of kisspeptin cell activation in both the ARC and mPOA.

Prenatal Testosterone Excess Decreases Neurokinin 3 Receptor Immunoreactivity within the Arcuate Nucleus KNDy Cell Population

Prenatal exposure of the female ovine foetus to excess testosterone leads to neuroendocrine disruptions in adulthood, as demonstrated by defects in responsiveness with respect to the ability of gonadal steroids to regulate gonadotrophin‐releasing hormone (GnRH) secretion. In the ewe, neurones of the arcuate nucleus (ARC), which co‐expresses kisspeptin, neurokinin B (NKB) and dynorphin (termed KNDy cells), play a key role in steroid feedback control of GnRH and show altered peptide expression after prenatal testosterone treatment. KNDy cells also co‐localise NKB receptors (NK3R), and it has been proposed that NKB may act as an autoregulatory transmitter in KNDy cells where it participates in the mechanisms underlying steroid negative‐feedback. In addition, recent evidence suggests that NKB/NK3R signalling may be involved in the positive‐feedback actions of oestradiol leading to the GnRH/luteinising hormone (LH) surge in the ewe. Thus, we hypothesise that decreased expression of NK3R in KNDy cells may be present in the brains of prenatal testosterone‐treated animals, potentially contributing to reproductive defects. Using single‐ and dual‐label immunohistochemistry we found NK3R‐positive cells in diverse areas of the hypothalamus; however, after prenatal testosterone treatment, decreased numbers of NK3R immunoreactive (‐IR) cells were seen only in the ARC. Moreover, dual‐label confocal analyses revealed a significant decrease in the percentage of KNDy cells (using kisspeptin as a marker) that co‐localised NK3R. To investigate how NKB ultimately affects GnRH secretion in the ewe, we examined GnRH neurones in the preoptic area (POA) and mediobasal hypothalamus (MBH) for the presence of NK3R. Although, consistent with earlier findings, we found no instances of NK3R co‐localisation in GnRH neurones in either the POA or MBH; in addition, > 70% GnRH neurones in both areas were contacted by NK3R‐IR presynaptic terminals suggesting that, in addition to its role at KNDy cell bodies, NKB may regulate GnRH neurones by presynaptic actions. In summary, the finding of decreased NK3R within KNDy cells in prenatal testosterone‐treated sheep complements previous observations of decreased NKB and dynorphin in the same population, and may contribute to deficits in the feedback control of GnRH/LH secretion in this animal model.

Expanding the role of tachykinins in the neuroendocrine control of reproduction

Reproductive function is driven by the hormonal interplay between the gonads and brain-pituitary axis. Gonadotropin-releasing hormone (GnRH) is released in a pulsatile manner, which is critical for the attainment and maintenance of fertility, however, GnRH neurons lack the ability to directly respond to most regulatory factors, and a hierarchical upstream neuronal network governs its secretion. We and others proposed a model in which Kiss1 neurons in the arcuate nucleus (ARC), so called KNDy neurons, release kisspeptin (a potent GnRH secretagogue) in a pulsatile manner to drive GnRH pulses under the coordinated autosynaptic action of its cotransmitters, the tachykinin neurokinin B (NKB, stimulatory) and dynorphin (inhibitory). Numerous genetic and pharmacological studies support this model; however, additional regulatory mechanisms (upstream of KNDy neurons) and alternative pathways of GnRH secretion (kisspeptin-independent) exist, but remain ill defined. In this aspect, attention to other members of the tachykinin family, namely substance P (SP) and neurokinin A (NKA), has recently been rekindled. Even though there are still major gaps in our knowledge about the functional significance of these systems, substantial evidence, as discussed below, is placing tachykinin signaling as an important pathway for the awakening of the reproductive axis and the onset of puberty to physiological GnRH secretion and maintenance of fertility in adulthood.

Activation of Cells Containing Estrogen Receptor Alpha or Somatostatin in the Medial Preoptic Area, Arcuate Nucleus, and Ventromedial Nucleus of Intact Ewes During the Follicular Phase, and Alteration after Lipopolysaccharide

ABSTRACT Cells in the medial preoptic area (mPOA), arcuate nucleus (ARC), and ventromedial nucleus (VMN) that possess estrogen receptor alpha (ER alpha) mediate estradiol feedback to regulate endocrine and behavioral events during the estrous cycle. A percentage of ER alpha cells located in the ARC and VMN express somatostatin (SST) and are activated in response to estradiol. The aims of the present study were to investigate the location of c-Fos, a marker for activation, in cells containing ER alpha or SST at various times during the follicular phase and to determine whether lipopolysaccharide (LPS) administration, which leads to disruption of the luteinizing hormone (LH) surge, is accompanied by altered ER alpha and/or SST activation patterns. Follicular phases were synchronized with progesterone vaginal pessaries, and control animals were killed at 0, 16, 31, and 40 h (n = 4–6/group) after progesterone withdrawal (PW [time 0]). At 28 h, other animals received LPS (100 ng/kg) and were subsequently killed at 31 h or 40 h (n = 5/group). Hypothalamic sections were immunostained for c-Fos and ER alpha or SST. LH surges occurred only in control ewes with onset at 36.7 ± 1.3 h after PW; these animals had a marked increase in the percentage of ER alpha cells that colocalized c-Fos (%ER alpha/c-Fos) in the ARC and mPOA from 31 h after PW and throughout the LH surge. In the VMN, %ER alpha/c-Fos was higher in animals that expressed sexual behavior than in those that did not. SST cell activation in the ARC and VMN was greater during the LH surge than in other stages in the follicular phase. At 31 or 40 h after PW (i.e., 3 or 12 h after treatment, respectively), LPS decreased %ER alpha/c-Fos in the ARC and the mPOA, but there was no change in the VMN compared to that in controls. The %SST/c-Fos increased in the VMN at 31 h after PW (i.e., 3 h after LPS) with no change in the ARC compared to controls. These results indicate that there is a distinct temporal pattern of ER alpha cell activation in the hypothalamus during the follicular phase, which begins in the ARC and mPOA at least 6–7 h before the LH surge onset and extends to the VMN after the onset of sexual behavior and LH surge. Furthermore, during the surge, some of these ER alpha-activated cells may be SST-secreting cells. This pattern is markedly altered by LPS administered during the late follicular phase, indicating that the disruptive effects of this stressor are mediated by suppressing ER alpha cell activation at the level of the mPOA and ARC and enhancing SST cell activation in the VMN, leading to the attenuation of the LH surge.

KNDy neurone activation prior to the LH surge of the ewe is disrupted by LPS

In the ewe, steroid hormones act on the hypothalamic arcuate nucleus (ARC) to initiate the GnRH/LH surge. Within the ARC, steroid signal transduction may be mediated by estrogen receptive dopamine-, β-endorphin- or neuropeptide Y (NPY)-expressing cells, as well as those co-localising kisspeptin, neurokinin B (NKB) and dynorphin (termed KNDy). We investigated the time during the follicular phase when these cells become activated (i.e., co-localise c-Fos) relative to the timing of the LH surge onset and may therefore be involved in the surge generating mechanism. Furthermore, we aimed to elucidate whether these activation patterns are altered after lipopolysaccharide (LPS) administration, which is known to inhibit the LH surge. Follicular phases of ewes were synchronised by progesterone withdrawal and blood samples were collected every 2 h. Hypothalamic tissue was retrieved at various times during the follicular phase with or without the administration of LPS (100 ng/kg). The percentage of activated dopamine cells decreased before the onset of sexual behaviour, whereas activation of β-endorphin decreased and NPY activation tended to increase during the LH surge. These patterns were not disturbed by LPS administration. Maximal co-expression of c-Fos in dynorphin immunoreactive neurons was observed earlier during the follicular phase, compared to kisspeptin and NKB, which were maximally activated during the surge. This indicates a distinct role for ARC dynorphin in the LH surge generation mechanism. Acute LPS decreased the percentage of activated dynorphin and kisspeptin immunoreactive cells. Thus, in the ovary-intact ewe, KNDy neurones are activated prior to the LH surge onset and this pattern is inhibited by the administration of LPS.

Co-expression of c-Fos with oestradiol receptor α or somatostatin in the arcuate nucleus, ventromedial nucleus and medial preoptic area in the follicular phase of intact ewes: alteration after insulin-induced hypoglycaemia.

The aim of this study was to investigate how acute insulin-induced hypoglycaemia (IIH) alters the activity of cells containing oestradiol receptor α (ERα) or somatostatin (SST) in the arcuate nucleus (ARC) and ventromedial nucleus (VMN), and ERα cells in the medial preoptic area (mPOA) of intact ewes. Follicular phases were synchronized with progesterone vaginal pessaries. Control animals were killed at 0 h or 31 h (n = 5 and 6, respectively) after progesterone withdrawal (PW; time zero). At 28 h, five other animals received insulin (INS; 4 iu/kg) and were subsequently killed at 31 h. Hypothalamic sections were immunostained for ERα or SST each with c-Fos, a marker of neuronal transcriptional activation. Insulin did not alter the percentage of activated ERα cells in the ARC; however, it appeared visually that two insulin-treated animals (INS responders, with no LH surge) had an increase in the VMN (from 32 to 78%) and a decrease in the mPOA (from 40 to 12%) compared to no increase in the two INS non-responders (with an LH surge). The percentage of activated SST cells in the ARC was greater in all four insulin-treated animals (from 10 to 60%), whereas it was visually estimated that activated SST cells in the VMN increased only in the two insulin responders (from 10 to 70%). From these results, we suggest that IIH stimulates SST activation in the ARC as part of the glucose-sensing mechanism but ERα activation is unaffected in this region. We present evidence to support a hypothesis that disruption of the GnRH/LH surge may occur in insulin responders via a mechanism that involves, at least in part, SST cell activation in the VMN along with decreased ERα cell activation in the mPOA.