In rodents, the ovulation-inducing luteinizing hormone (LH) surge is sexually dimorphic, occurring only in females, but the reasons for this sex difference are unclear. Two neuropeptides, kisspeptin and RFamide-related peptide 3 (RFRP-3), are hypothesized to regulate the gonadotropin-releasing hormone (GnRH)/LH surge. In females, both of these systems show circadian changes coincident with the LH surge, but whether males show similar temporal changes under comparable hormonal conditions is unknown. Here, we evaluated circadian time (CT)-dependent changes in gene expression and neuronal activation of Kiss1 and Rfrp neurons of female and male mice given identical LH surge-inducing estrogen regimens. As expected, females, but not males, displayed a late afternoon LH surge and GnRH neuronal activation. Kiss1 expression in the anteroventral periventricular nucleus (AVPV) was temporally increased in females in the late afternoon, whereas males demonstrated no temporal changes in AVPV Kiss1 expression. Likewise, neuronal activation of AVPV Kiss1 neurons was dramatically elevated in the late afternoon in females but was low at all circadian times in males. Estrogen receptor a levels in AVPV Kiss1 neurons were sexually dimorphic, being higher in females than males. AVPV progesterone receptor levels were also higher in females than males. Hypothalamic Rfrp messenger RNA levels showed no CT-dependent changes in either sex. However, Rfrp neuronal activation was temporally diminished in the afternoon/evening in females but not males. Collectively, the identified sex differences in absolute and CT-dependent AVPV Kiss1 levels, AVPV sex steroid receptor levels, and circadian-timed changes in neuronal activation of both Kiss1 and Rfrp neurons suggest that multiple sexually dimorphic processes in the brain may underlie proper LH surge generation.

Polycystic ovary syndrome (PCOS) pathophysiology is poorly understood, due partly to lack of PCOS animal models fully recapitulating this complex disorder. Recently, a PCOS rat model using letrozole (LET), a nonsteroidal aromatase inhibitor, mimicked multiple PCOS phenotypes, including metabolic features absent in other models. Given the advantages of using genetic and transgenic mouse models, we investigated whether LET produces a similar PCOS phenotype in mice. Pubertal female C57BL/6N mice were treated for 5 wk with LET, which resulted in increased serum testosterone and normal diestrus levels of estradiol, similar to the hyperandrogenemia and follicular phase estrogen levels of PCOS women. As in PCOS, ovaries from LET mice were larger, polycystic, and lacked corpora lutea versus controls. Most LET females were acyclic, and all were infertile. LET females displayed elevated serum LH levels and higher Lhb mRNA in the pituitary. In contrast, serum FSH and Fshb were significantly reduced in LET females, demonstrating differential effects on gonadotropins, as in PCOS. Within the ovary, LET females had higher Cyp17, Cyp19, and Fsh receptor mRNA expression. In the hypothalamus, LET females had higher kisspeptin receptor mRNA expression but lower progesterone receptor mRNA levels. LET females also gained more weight than controls, had increased abdominal adiposity and adipocyte size, elevated adipose inflammatory mRNA levels, and impaired glucose tolerance, mirroring the metabolic phenotype in PCOS women. This is the first report of a LET paradigm in mice that recapitulates both reproductive and metabolic PCOS phenotypes and will be useful to genetically probe the PCOS condition.

Kisspeptin, encoded by Kiss1, stimulates GnRH neurons to govern reproduction. In rodents, estrogen-sensitive kisspeptin neurons in the anterior ventral periventricular nucleus and neighboring periventricular nucleus are thought to mediate sex steroid-induced positive feedback induction of the preovulatory LH surge. These kisspeptin neurons coexpress estrogen and progesterone receptors and display enhanced neuronal activation during the LH surge. However, although estrogen regulation of kisspeptin neurons has been well studied, the role of progesterone signaling in regulating kisspeptin neurons is unknown. Here we tested whether progesterone action specifically in kisspeptin cells is essential for proper LH surge and fertility. We used Cre-lox technology to generate transgenic mice lacking progesterone receptors exclusively in kisspeptin cells (termed KissPRKOs). Male KissPRKOs displayed normal fertility and gonadotropin levels. In stark contrast, female KissPRKOs displayed earlier puberty onset and significant impairments in fertility, evidenced by fewer births and substantially reduced litter size. KissPRKOs also had fewer ovarian corpora lutea, suggesting impaired ovulation. To ascertain whether this reflects a defect in the ability to generate sex steroid-induced LH surges, females were exposed to an estradiol-positive feedback paradigm. Unlike control females, which displayed robust LH surges, KissPRKO females did not generate notable LH surges and expressed significantly blunted cfos induction in anterior ventral periventricular nucleus kisspeptin neurons, indicating that progesterone receptor signaling in kisspeptin neurons is required for normal kisspeptin neuronal activation and LH surges during positive feedback. Our novel findings demonstrate that progesterone signaling specifically in kisspeptin cells is essential for the positive feedback induction of normal LH surges, ovulation, and normal fertility in females.

Throughout most of the ovulatory cycle, estrogen negative feedback restrains the GnRH neuronal system. Just before ovulation, however, estrogen negative feedback is removed to permit stimulation of the preovulatory GnRH/LH surge (positive feedback) by the circadian clock in the suprachiasmatic nucleus (SCN). The mammalian ortholog of avian gonadotropin-inhibitory hormone, RFamide-related peptide 3 (RFRP-3), participates in the circadian-timed removal of estrogen negative feedback to permit the LH surge. The present study examined the specific neurochemical means by which the SCN controls RFRP-3 activity and explored whether the RFRP-3 system exhibits time-dependent responsiveness to SCN signaling to precisely time the LH surge. We found that RFRP-3 cells in female Syrian hamsters (Mesocricetus auratus) receive close appositions from SCN-derived vasopressin-ergic and vasoactive intestinal peptide (VIP)-ergic terminal fibers. Central VIP administration markedly suppressed RFRP-3 cellular activity in the evening, but not the morning, relative to saline controls, whereas vasopressin was without effect at either time point. Double-label in situ hybridization for Rfrp-3 and the VIP receptors VPAC1 and VPAC2 revealed that the majority of RFRP-3 cells do not coexpress either receptor in Syrian hamsters or mice, suggesting that SCN VIP-ergic signaling inhibits RFRP-3 cells indirectly. The timing of this VIP-mediated disinhibition is further coordinated via temporally gated responsiveness of RFRP-3 cells to circadian signaling. Together, these findings reveal a novel circadian hierarchy of control coordinating the preovulatory LH surge and ovulation.

RFamide-related peptide-3 (RFRP-3) [mammalian ortholog to gonadotropin-inhibiting hormone (GnIH)] potently inhibits gonadotropin secretion in mammals. Studies of RFRP-3 immunoreactivity and Rfrp expression (the gene encoding RFRP-3) in mammalian brains have uncovered several possible pathways regulating RFRP-3 neurons, shedding light on their potential role in reproduction and other processes, and pharmacological studies have probed the target sites of RFRP-3 action. Despite this, there is currently no major consensus on RFRP-3's specific endogenous role(s) in reproductive physiology. Here, we discuss the latest evidence relating to RFRP-3 neuron regulation and function during development and sexual maturation, focusing on rodents. We highlight significant changes in RFRP-3 and Rfrp expression, as well as RFRP-3 neuronal activation, during key stages of postnatal and pubertal development and also discuss recent evidence testing the requisite role of RFRP-3 receptors for normal pubertal timing and developmental LH secretion. Interestingly, some findings suggest that endogenous RFRP-3 signaling may not be necessary for the puberty timing, at least in some species, forcing new hypotheses to be generated regarding this peptide's functional significance to sexual maturation and development.

I.J. Clarke, Clayton, Vic. C. Coen, London M.A. Cowley, Clayton, Vic. W.W. de Herder, Rotterdam S.L. Dickson, Gothenburg A. Enjalbert, Marseille A.B. Grossman, Headington D. Jezová, Bratislava A.S. Kauff man, La Jolla, Calif. M.J. Kelly, Portland, Oreg. P.A. Kelly, Paris X. Ni, Shanghai S.R. Ojeda, Beaverton, Oreg. (Reviews) G. Rindi, Rome H. Sasano, Sendai J.Y. Seong, Seoul M. Tena-Sempere, Cordoba H. Tsukamura, Nagoya E.J. Wagner, Pomona, Calif. B. Wiedenmann, Berlin Z. Zeng, Beijing Editorial Board

Background: The neuropeptide RFamide-related peptide-3 (RFRP-3; mammalian ortholog to gonadotropin-inhibiting hormone) can inhibit luteinizing hormone (LH) release and increases feeding, but the regulation and development of RFRP-3 neurons remains poorly characterized, especially in mice. Methods and Results: We first confirmed that peripheral injections of murine RFRP-3 peptide could markedly suppress LH secretion in adult mice, as in other species. Second, given RFRP-3's reported orexigenic properties, we performed double-label in situ hybridization for metabolic genes in Rfrp neurons of mice. While Rfrp neurons did not readily coexpress neuropeptide Y, thyrotropin-releasing hormone, or MC4R, a small subset of Rfrp neurons did express the leptin receptor in both sexes. Surprisingly, we identified no changes in Rfrp expression or neuronal activation in adult mice after acute fasting. However, we determined that Rfrp mRNA levels in the dorsal-medial nucleus were significantly reduced in adult obese (Ob) mice of both sexes. Given the lower Rfrp levels observed in adult Ob mice, we asked whether leptin might also regulate RFRP-3 neuron development. Rfrp gene expression changed markedly over juvenile development, correlating with the timing of the juvenile ‘leptin surge' known to govern hypothalamic feeding circuit development. However, the dramatic developmental changes in juvenile Rfrp expression did not appear to be leptin driven, as the pattern and timing of Rfrp neuron development were unaltered in Ob juveniles. Conclusion: Leptin status modulates RFRP-3 expression in adulthood, but is not required for normal development of the RFRP-3 system. Leptin's regulation of adult RFRP-3 neurons likely occurs primarily via indirect signaling, and may be secondary to obesity, as only a small subset of RFRP-3 neurons express the long form of the leptin receptor (LepRb).

Kisspeptin, encoded by the Kiss1 gene, is a neuropeptide required for puberty and adult reproductive function. Understanding the regulation and development of the kisspeptin system provides valuable knowledge about the physiology of puberty and adult fertility, and may provide insights into human pubertal or reproductive disorders. Recent studies, particularly in rodent models, have assessed how kisspeptin neurons develop and how hormonal and non-hormonal factors regulate this developmental process. Exposure to sex steroids (testosterone and estradiol) during critical periods of development can induce organizational (permanent) effects on kisspeptin neuron development, with respect to both sexually dimorphic and non-sexually dimorphic aspects of kisspeptin biology. In addition, sex steroids can also impart activational (temporary) effects on kisspeptin neurons and Kiss1 gene expression at various times during neonatal and peripubertal development, as they do in adulthood. Here, we discuss the current knowledge--and in some cases, lack thereof--of the influence of hormones and other factors on kisspeptin neuronal development.

Arginine-phenylalanine-amide (RFamide)-related peptide 3 (RFRP-3, encoded by the Rfrp gene) is the mammalian ortholog of gonadotropin-inhibiting hormone and can inhibit GnRH neuronal activity and LH release. However, the development and regulation of the RFRP-3 system in both sexes is poorly understood. Using in situ hybridization, we examined changes in Rfrp-expressing neurons in mice of both sexes during development and under different adulthood hormonal milieus. We found no sex differences in Rfrp expression or cell number in adult mice. Interestingly, we identified two interspersed subpopulations of Rfrp cells (high Rfrp-expressing, HE; low Rfrp-expressing, LE), which have unique developmental and steroidal regulation characteristics. The number of LE cells robustly decreases during postnatal development, whereas HE cell number increases significantly before puberty. Using Bax knockout mice, we determined that the dramatic developmental decrease in LE Rfrp cells is not due primarily to BAX-dependent apoptosis. In adults, we found that estradiol and testosterone moderately repress Rfrp expression in both HE and LE cells, whereas the nonaromatizable androgen dihydrotestosterone has no effect. Using double-label in situ hybridization, we determined that approximately 25% of Rfrp neurons coexpress estrogen receptor-α in each sex, whereas Rfrp cells do not readily express androgen receptor in either sex, regardless of hormonal milieu. Lastly, when we looked at RFRP-3 receptors, we detected some coexpression of Gpr147 but no coexpression of Gpr74 in GnRH neurons of both intact and gonadectomized males and females. Thus, RFRP-3 may exert its effects on reproduction either directly, via Gpr147 in a subset of GnRH neurons, and/or indirectly, via upstream regulators of GnRH.

RFamide-related peptide-3 (RFRP-3) is known to inhibit the activity of GnRH neurons. It is not yet clear whether its G protein-coupled receptors, GPR147 and GPR74, are present on GnRH neurons or on afferent inputs of the GnRH neuronal network or whether RFRP-3 can inhibit gonadotropin secretion independently of GnRH. We tested the following: 1) whether GnRH is essential for the effects of RFRP-3 on LH secretion; 2) whether RFRP-3 neurons project to GnRH and rostral periventricular kisspeptin neurons in mice, and 3) whether Gpr147 and Gpr74 are expressed by these neurons. Intravenous treatment with the GPR147 antagonist RF9 increased plasma LH concentration in castrated male rats but was unable to do so in the presence of the GnRH antagonist cetrorelix. Dual-label immunohistochemistry revealed that approximately 26% of GnRH neurons from male and diestrous female mice were apposed by RFRP-3 fibers, and 19% of kisspeptin neurons from proestrous female mice were apposed by RFRP-3 fibers. Using immunomagnetic purification of GnRH and kisspeptin cells, single-cell nested RT-PCR, and in situ hybridization, we showed that 33% of GnRH neurons and 9-16% of rostral periventricular kisspeptin neurons expressed Gpr147, whereas Gpr74 was not expressed in either population. These data reveal that RFRP-3 can act at two levels of the GnRH neuronal network (i.e. the GnRH neurons and the rostral periventricular kisspeptin neurons) to modulate reproduction but is unable to inhibit gonadotropin secretion independently of GnRH.

Kisspeptin, a neuropeptide encoded by the Kiss1 gene, is a potent secretagogue of GnRH, and is therefore critical for sex steroid secretion in adulthood. However, kisspeptin's role in regulating sex steroid secretion during perinatal development is unexplored. In mice, testosterone (T) levels are higher in males than females several days before birth and again later during the first five hours after birth (postnatal day 1). Evidence suggests that prenatal gonadal T secretion may occur independently of GnRH influence; however, whether the elevated T in postnatal day 1 (P1) male mice is governed by GnRH neurons or upstream kisspeptin circuits is unknown. In Exp. 1, we determined the role of kisspeptin-Kiss1r signaling in P1 T secretion. Blood and brains were collected either within the first 4 hours after birth ("4h" group; when T is normally elevated in males but not females) or 16-20 hours after birth ("20h" group) in P1 Kiss1r KO and wildtype (WT) mice. First, we used in situ hybridization to determine if higher T levels in 4h WT males than females might be driven by higher upstream Kiss1 levels in the brains of males. Interestingly, there was no difference in hypothalamic Kiss1 expression between 4h and 20h males, and males did not have higher Kiss1 levels than females at either time-point. Next, we measured serum T levels in P1 mice of both genotypes. As expected, 4h WT males had higher T levels than 4h WT females. Surprisingly, 4h Kiss1r KO males also had higher T levels than 4h females, indicating that P1 males retain their ability to secrete T in the absence of kisspeptin signaling. Next, in Exp. 2, we tested whether kisspeptin-Kiss1r signaling is required for prenatal T secretion. We used in situ hybridization to examine hypothalamic progesterone receptor (Pgr) expression, a well-established proxy for T levels in prenatal rodents (higher T levels in embryonic males promote hypothalamic Pgr expression). Interestingly, we found no difference in hypothalamic Pgr expression between embryonic day 18 (E18) Kiss1r KO and WT males, both of which had much higher Pgr levels than E18 females. This suggests that Kiss1r KO males retain the ability to secrete elevated T during late embryonic life. We conclude that, unlike in adulthood, elevated prenatal and postnatal T secretion in mice is likely independent of kisspeptin signaling. We are currently investigating if perinatal T secretion is also independent of GnRH signaling using hpg mutant mice. Collectively, our results will help address whether the perinatal testis' endocrine function is dependant or independent of hypothalamic regulation. (platform)

Kisspeptin, encoded by the Kiss1 gene, stimulates GnRH secretion and is therefore critical for sex steroid secretion at puberty and in adulthood. However, kisspeptin's role in regulating sex steroid secretion earlier in development is unexplored. In rodents, testosterone (T) levels are higher in prenatal and newborn males than females. We determined whether kisspeptin-Kiss1r and GnRH signaling plays a role in sexually dimorphic perinatal T secretion in mice. Our results demonstrate that 1) T levels in newborn males are elevated at 4 h but not 20 h after birth, but hypothalamic Kiss1 and neurokinin B (NKB) levels in males are not different between these time points (and both are lower than in females); 2) serum T levels in newborn Kiss1r knockout (KO) males are higher than in newborn females and similar to wild-type (WT) males; 3) perinatal hypothalamic progesterone receptor (Pgr) expression, which is dependent on circulating levels of gonadally produced T, is significantly higher in prenatal and newborn Kiss1r KO and WT males than similarly aged females; 4) multiple measures of testicular growth and function are not different between developing Kiss1r KO and WT mice until after postnatal d 5; and 5) GnRH neurons of newborn males do not exhibit high c-fos coexpression, and newborn hypogonadal (hpg) male mice (lacking GnRH) secrete elevated T, similar to newborn WT males. We conclude that, unlike in puberty and adulthood, elevated T secretion in prenatal and neonatal mice is independent of both kisspeptin and GnRH signaling, and the necessity of kisspeptin-Kiss1r signaling for testicular function is first apparent after d 5.

The Kiss1 gene and its product kisspeptin are important regulators of reproduction. In rodents, Kiss1 is expressed in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV)/rostral periventricular (PeN) nuclei. In the AVPV/PeN, females have more Kiss1 and tyrosine hydroxylase (TH) neurons than males. We explored the ontogeny of the Kiss1 sex difference, and the role of cell death in establishing Kiss1 and TH cell number. We also determined whether Kiss1 cells in AVPV/PeN coexpress TH. AVPV/PeN Kiss1 neurons were first detected in both sexes on postnatal d 10, but the Kiss1 sex difference did not emerge until postnatal d 12. The role of BAX-mediated apoptosis in generating this sex difference was tested in adult Bax knockout (KO) and wild-type mice. Deletion of Bax did not diminish the sex difference in Kiss1 expression in the AVPV/PeN. TH expression was sexually dimorphic in the AVPV of both wild-type and Bax KO mice but, unlike Kiss1, was not sexually dimorphic in the PeN of either genotype. Double-label analysis determined that most Kiss1 neurons coexpress TH mRNA, but many TH neurons do not coexpress Kiss1, especially in the PeN. These findings suggest that several subpopulations of TH cells reside within the AVPV/PeN, only one of which coexpresses Kiss1. In the ARC, Kiss1 cell number was markedly increased in Bax KO mice of both sexes, indicating that although BAX-dependent apoptosis does not generate the sex difference in either Kiss1 or TH expression in AVPV/PeN, BAX does importantly regulate Kiss1 cell number in the ARC.

Interspecific (xeno) transfer of embryonic primordial germ cells (PGCs) from quail (Coturnix japonica) to chicken (Gallus gallus) has produced germ cell chimeras that generate viable donor-derived germ cells. The practical application of this technique for the preservation of endangered species requires the use of gonadal stem cells (GSCs) recovered from adult birds at necropsy. Using the quail to chicken model, we examined the feasibility and efficacy of isolating adult GSCs for xenotransfer into host embryos. In this study, GSCs from adult quail testes were quantified by flow cytometry using antibodies against GSC-specific antigens: stage-specific embryonic antigen-1 (SSEA1; 88%), stage-specific embryonic antigen-4 (SSEA4; <1%), CD9; <1%, epithelial membrane antigen-1 (EMA1; <1%), integrin alpha-6 (alpha6; <1%), and integrin beta-1 (beta1; 3%). The high percentage of SSEA1 positive cells indicates that this antigen is too widely expressed to be a differential stem cell marker for adult testicular cells. The percentage of beta1 positive cells suggests that this marker is more suitable for stem cell identification in this model. GSC populations were then enriched by magnetic activated cell sorting (MACS). Following MACS, the proportion of testicular cells expressing stem cell antigens increased: SSEA1, 1.1 fold; SSEA4, 40.3; EMA1, 70.1; CD9, 209.7: alpha6, 114.6; beta1, 23.4. Finally, we quantified the expression of stem cell markers in the embryonic gonad of 11-day incubated chicken embryos to determine the characteristics of control, non-chimeric host tissue for comparison to post-xenotransfer chimeric gonads. In the male embryonic gonad, we found a greater number of SSEA1 and β1 antigen expressing cells and low expression levels of the other antigens tested (SSEA4, CD9, EMA1, alpha6). We found a similar pattern in the female embryonic gonad, with a SSEA1 and β1 expressed at higher levels than the other antigens tested. Ideally, we aim to transplant a GSC enriched population coincident with endogenous PGC migration that will best integrate into the host gonad and outnumber, thus supplant, the endogenous PGCs. The quail to chicken germ cell chimera using adult gonadal tissue is a model for the development of xenotransfer of endangered avian GSCs. With this technique, GSC rescued from genetically important or endangered species may survive to produce functional gametes in the gonads of non-endangered hosts. (poster)

Conservation of germplasm from endangered and genetically valuable individuals is a high priority for all classes of animals. However, the large avian oocyte and the inability to consistently super ovulate birds make techniques such as cloning and oocyte cryopreservation unlikely tools for avian conservation. Instead, the use of domestic birds as hosts to produce sperm of exotic species for use in artificial insemination may be a practical approach to conserve avian germplasm. During early embryonic development the primordial germ stem cells (PGCs) circulate in the blood vessels prior to colonization of the gonadal ridge. These circulating PGCs are the precursors for the adult germ stem cells (GSCs), which allow continual production of sperm throughout the lifetime of males. Previous studies have demonstrated the usefulness of embryonic PGC transfers for preserving rare chicken lines, introducing transgenes, or xenotransfer between chicken, quail and turkeys. We propose that the use of non-seasonal domesticated hosts (e.g. chickens or quail) can ensure year-round production of genetically valuable sperm for use in assisted reproductive technologies such as artificial insemination. In this study adult quail testes were dispersed with collagenase prior to injection into the viteline vein of stage 15-18 chicken embryos coincident with endogenous PGC migration. Host chicken gonads were collected at day 8 of incubation and collagenase dispersed. Donor (quail) derived cells were detected by the quail specific antibody QH1 and counted by flowcytometry. Up to 2.3% of chicken embryo gonadal cells were positive for QH1 and presumably derived from the quail donor testis. This model system will be used to develop techniques for isolation and xenotransfer of GSCs collected during necropsy of adult birds, thereby enhancing the preservation of genetic material from endangered or genetically valuable specimens. (poster)

Chronic viral infections kill millions of people annually worldwide. Loss of T cell function has been noticed for long time during chronic infections but the underlying factors are not completely understood. Here, we evaluate the role of TGF‐β in the establishment of a persistent viral infection. For that, we infected mice expressing a dominant negative form of TGF‐β receptor II from a modified CD4 promoter (dnTGFBRII) with a persistent variant of lymphocytic choriomeningitis virus, Clone 13 (Cl 13). We found that attenuated TGF‐β signaling in T cells allows the expansion of virus specific CD8 T cells in Cl 13 infected mice resulting in potent cytokine production, enhanced cytotoxic capacity and down‐regulated levels of προγραμμεδ χελλ δεατη−1. Μορεοϖε, ιν χοντραστ το τηειρ ωιλδ τψπε χουντερπαρτσ, Χλ 13−ινφεχτεδ−δνΤΓΦΒΡΙΙ μιχε ραπιδλψ ελιμινατε τηε ϖιρυσ φρο μ βλοοδ ανδ σεϖ εραλ τισσυεσ. Ουρ δατα ιδεντιφψ α Τ χελλ−ιντρινσιχ ινηιβιτο ρψ πατηωαψ ρεσπονσιβλε φορ ΧΔ8 Τ χελλ εξηαυστιον ανδ ϖιραλ περσιστενχε ινδιχατινγ τηατ σελεχτιϖε μο δυλατιον οφ ΤΓΦ−β signaling on T cells holds a strong promise for alternative treatments of chronic viral infections.