The short reproductive cycle length observed in rodents, called the estrous cycle, makes them an ideal animal model for investigation of changes that occur during the reproductive cycle. Most of the data in the literature about the estrous cycle is obtained from rat because they are easily manipulated and exhibit a clear and well defined estrous cycle. However, the increased number of experiments using knockout mice requires identification of their estrous cycle as well, since (in)fertility issues may arise. In mice, like rats, the identification of the stage of estrous cycle is based on the proportion of cells types observed in the vaginal secretion. The aim of this unit is to provide guidelines for quickly and accurately determining estrous cycle phases in mice. Key terms for indexing Estrous cycle; mouse; vaginal smears; wet vaginalIn humans, the reproductive cycle, called the menstrual cycle, lasts approximately 28 days, in rodents this cycle, called the estrous cycle, lasts approximately 4-5 days. The success of reproduction in all mammals depends on the function of the hypothalamus-pituitary-gonads axis. In females, gonadotropin-releasing hormone (GnRH) neurons present in the septal area and hypothalamus send their axons to median eminence. GnRH released there reaches the anterior pituitary where the gonadotrophs are stimulated to secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In the peripheral circulation, these hormones stimulate specific cells in the ovary leading to ovulation. Ovarian follicles secrete estrogen (E2) that reaches the hypothalamus. Most of the time, the plasma concentration of E2 is low. However, during the pre-ovulatory period E2 secretion reaches a peak, and GnRH neurons are stimulated (Sarkar et al., 1976;Freeman, 1994). E2 seems to regulate GnRH neurons indirectly, via interneurons. However, there are many questions needing to be answered in this field. Importantly, because E2 has a myriad of effects, questions are raised not only in the neuroendocrine field, but also in the neurosciences (e.g. synaptic plasticity, memory and learning and neurodegenerative diseases) as well as fields studying cardiological and/or renal function, sodium and water equilibrium, etc.A short cycle length makes rodents an ideal animal model for investigating changes occurring during the reproductive cycle and historically rats have been the chosen model. Rats display, most of time, regular cycles; they are easy to manipulate; and the cycle is not disrupted easily even with the routine stress in the animal facility. However, as use of mice lines continues to increase, an understanding of the mouse estrous cycle is critical for investigators and in fact many knockout mice can exhibit puberty/fertility changes that can effect, simply, maintaining a knockout line. There are few published studies involving estrous cycle in mice. The stages of the estrous cycle are not as visually discernible as in rats, and handling mice requires more caution due to their aggressive behavior. M...

Kisspeptins, the natural ligands of the G-protein-coupled receptor (GPR)-54, are the most potent stimulators of GnRH-1 secretion and as such are critical to reproductive function. However, the mechanism by which kisspeptins enhance calcium-regulated neuropeptide secretion is not clear. In the present study, we used GnRH-1 neurons maintained in mice nasal explants to examine the expression and signaling of GPR54. Under basal conditions, GnRH-1 cells exhibited spontaneous baseline oscillations in intracellular calcium concentration ([Ca(2+)](i)), which were critically dependent on the operation of voltage-gated, tetrodotoxin (TTX)-sensitive sodium channels and were not coupled to calcium release from intracellular pools. Activation of native GPR54 by kisspeptin-10 initiated [Ca(2+)](i) oscillations in quiescent GnRH-1 cells, increased the frequency of calcium spiking in oscillating cells that led to summation of individual spikes into plateau-bursting type of calcium signals in a subset of active cells. These changes predominantly reflected the stimulatory effect of GPR54 activation on the plasma membrane oscillator activity via coupling of this receptor to phospholipase C signaling pathways. Both components of this pathway, inositol 1,3,4-trisphosphate and protein kinase C, contributed to the receptor-mediated modulation of baseline [Ca(2+)](i) oscillations. TTX and 2-aminoethyl diphenylborinate together abolished agonist-induced elevation in [Ca(2+)](i) in almost all cells, whereas flufenamic acid was less effective. Together these results indicate that a plasma membrane calcium oscillator is spontaneously operative in the majority of prenatal GnRH-1 neurons and is facilitated by kisspeptin-10 through phosphatidyl inositol diphosphate hydrolysis and depolarization of neurons by activating TTX-sensitive sodium channels and nonselective cationic channels.

The hypothalamic kisspeptin signaling system is a major positive regulator of the reproductive neuroendocrine axis, and loss of Kiss1 in the mouse results in infertility, a condition generally attributed to its hypogonadotropic hypogonadism. We demonstrate that in Kiss1(-/-) female mice, acute replacement of gonadotropins and estradiol restores ovulation, mating, and fertilization; however, these mice are still unable to achieve pregnancy because embryos fail to implant. Progesterone treatment did not overcome this defect. Kiss1(+/-) embryos transferred to a wild-type female mouse can successfully implant, demonstrating the defect is due to maternal factors. Kisspeptin and its receptor are expressed in the mouse uterus, and we suggest that it is the absence of uterine kisspeptin signaling that underlies the implantation failure. This absence, however, does not prevent the closure of the uterine implantation chamber, proper alignment of the embryo, and the ability of the uterus to undergo decidualization. Instead, the loss of Kiss1 expression specifically disrupts embryo attachment to the uterus. We observed that on the day of implantation, leukemia inhibitory factor (Lif), a cytokine that is absolutely required for implantation in mice, is weakly expressed in Kiss1(-/-) uterine glands and that the administration of exogenous Lif to hormone-primed Kiss1(-/-) female mice is sufficient to partially rescue implantation. Taken together, our study reveals that uterine kisspeptin signaling regulates glandular Lif levels, thereby identifying a novel and critical role for kisspeptin in regulating embryo implantation in the mouse. This study provides compelling reasons to explore this role in other species, particularly livestock and humans.

Patients bearing mutations in TAC3 and TACR3 (which encode neurokinin B and its receptor, respectively) have sexual infantilism and infertility due to GnRH deficiency. In contrast, Tacr3(-/-) mice have previously been reported to be fertile. Because of this apparent phenotypic discordance between mice and men bearing disabling mutations in Tacr3/TACR3, Tacr3 null mice were phenotyped with close attention to pubertal development, estrous cyclicity, and fertility. Tacr3(-/-) mice demonstrated normal timing of preputial separation and day of first estrus, markers of sexual maturation. However, at postnatal d 60, Tacr3(-/-) males had significantly smaller testes and lower FSH levels than their wild-type littermates. Tacr3(-/-) females had lower uterine weights and abnormal estrous cyclicity. Approximately half of Tacr3(-/-) females had no detectable corpora lutea on ovarian histology at postnatal d 60. Despite this apparent ovulatory defect, all Tacr3(-/-) females achieved fertility when mated. However, Tacr3(-/-) females were subfertile, having both reduced numbers of litters and pups per litter. The subfertility of these animals was not due to a primary ovarian defect, because they demonstrated a robust response to exogenous gonadotropins. Thus, although capable of fertility, Tacr3-deficient mice have central reproductive defects. The remarkable ability of acyclic female Tacr3 null mice to achieve fertility is reminiscent of the reversal of hypogonadotropic hypogonadism seen in a high proportion of human patients bearing mutations in TACR3. Tacr3 mice are a useful model to examine the mechanisms by which neurokinin B signaling modulates GnRH release.

Kisspeptin (Kiss1) signaling to GnRH neurons is widely acknowledged to be a prerequisite for puberty and reproduction. Animals lacking functional genes for either kisspeptin or its receptor exhibit low gonadotropin secretion and infertility. Paradoxically, a recent study reported that genetic ablation of nearly all Kiss1-expressing neurons (Kiss1 neurons) does not impair reproduction, arguing that neither Kiss1 neurons nor their products are essential for sexual maturation. We posited that only minute quantities of kisspeptin are sufficient to support reproduction. If this were the case, animals having dramatically reduced Kiss1 expression might retain fertility, testifying to the redundancy of Kiss1 neurons and their products. To test this hypothesis and to determine whether males and females differ in the required amount of kisspeptin needed for reproduction, we used a mouse (Kiss1-CreGFP) that has a severe reduction in Kiss1 expression. Mice that are heterozygous and homozygous for this allele (Kiss1(Cre/+) and Kiss1(Cre/Cre)) have ∼50% and 95% reductions in Kiss1 transcript, respectively. We found that although male Kiss1(Cre/Cre) mice sire normal-sized litters, female Kiss1(Cre/Cre) mice exhibit significantly impaired fertility and ovulation. These observations suggest that males require only 5% of normal Kiss1 expression to be reproductively competent, whereas females require higher levels for reproductive success.

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Gonadotrophin-releasing hormone (GnRH) neurones constitute the final output pathway of a neuronal network that controls the preovulatory luteinising hormone (LH) surge and ovulation. Throughout the reproductive cycle, several neurotransmitters stimulate and inhibit the activity of GnRH neurones, including oxytocin. The central administration of oxytocin antiserum abolishes the pro-oestrous LH surge whereas oxytocin stimulates GnRH secretion from hypothalamic explants suggesting an oxytocin central action. Within the GnRH neuronal population in the rat, GnRH cells in the medial preoptic area (MPOA) are activated at the time of the LH surge. Thus, we hypothesised that GnRH neurones in the MPOA may express oxytocin receptors, and that oxytocin neurones in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) may be differentially activated during the oestrous cycle. Oxytocin receptors mRNA was detected in the MPOA using reverse transcription-polymerase chain reaction. In animals in either metoestrus or pro-oestrus, double-label immunofluorescence indicated that approximately 10% of GnRH neurones in the MPOA coexpressed oxytocin receptors and that a few oxytocin fibres are located in the vicinity of these GnRH neurones. However, other neurones positive for the oxytocin receptors were found near GnRH neurones. At both oestrous stages, double-label immunofluorescence revealed that approximately 30% of oxytocin neurones in the SON were Fos-positive whereas oxytocin neurones in the PVN were consistently Fos-negative. Together, these data suggest that oxytocin may directly control neuronal activity in a subpopulation of GnRH neurones. Moreover, both oxytocin neuronal activity and the oxytocin receptor expression on GnRH cells are not influenced by oestrogen.

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