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Reproductive function is suppressed during lactation owing to the suckling-induced suppression of the kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and subsequent suppression of luteinizing hormone (LH) release. Our previous study revealed that somatostatin (SST) neurons mediate suckling-induced suppression of LH release via SST receptor 2 (SSTR2) in ovariectomized lactating rats during early lactation. This study examined whether central SST-SSTR2 signaling mediates the inhibition of ARC Kiss1 expression and LH release in lactating rats during late lactation and whether the inhibition of glutamatergic neurons, stimulators of LH release, is involved in the suppression of LH release mediated by central SST-SSTR2 signaling in lactating rats. A central injection of the SSTR2 antagonist CYN154806 (CYN) significantly increased ARC Kiss1 expression in lactating rats on day 16 of lactation. Dual in situ hybridization revealed that few ARC Kiss1-positive cells co-expressed Sstr2, and some of the ARC Slc17a6 (a glutamatergic neuronal marker)-positive cells co-expressed Sstr2. Furthermore, almost all ARC Kiss1-positive cells co-expressed Grin1, a subunit of N-methyl-D-aspartate (NMDA) receptors. The numbers of Slc17a6/Sstr2 double-labeled and Slc17a6 single-labeled cells were significantly lower in lactating dams than in non-lactating rats whose pups had been removed after parturition. A central injection of an NMDA antagonist reversed the CYN-induced increase in LH release in lactating rats. Overall, these results suggest that central SST-SSTR2 signaling, at least partly, mediates the suppression of ARC Kiss1 expression and LH release by inhibiting ARC glutamatergic interneurons in lactating rats.

Accumulating evidence suggests that kisspeptin-GPR54 signaling is indispensable for gonadotropin-releasing hormone (GnRH)/gonadotropin secretion and consequent reproductive functions in mammals. Conventional Kiss1 knockout (KO) mice and rats are reported to be infertile. To date, however, no study has investigated the effect of inducible central Kiss1 KO/knockdown on pulsatile gonadotropin release in male mammals. Here we report an in vivo analysis of inducible conditional Kiss1 knockdown male mice. The mice were generated by a bilateral injections of either adeno-associated virus (AAV) vectors driving Cre recombinase (AAV-Cre) or AAV vectors driving GFP (AAV-GFP, control) into the hypothalamic arcuate nucleus (ARC) of Kiss1-floxed male mice, in which exon 3 of the Kiss1 gene were floxed with loxP sites. Four weeks after the AAV-Cre injection, the mice showed a profound decrease in the both number of ARC Kiss1-expressing cells and the luteinizing hormone (LH) pulse frequency. Interestingly, pulsatile LH secretion was apparent 8 weeks after the AAV-Cre injection despite the suppression of ARC Kiss1 expression. The control Kiss1-floxed mice infected with AAV-GFP showed apparent LH pulses and Kiss1 expression in the ARC at both 4 and 8 weeks after the AAV-GFP injection. These results with an inducible conditional Kiss1 knockdown in the ARC of male mice suggest that ARC kisspeptin neurons are responsible for pulsatile LH secretion in male mice, and indicate the possibility of a compensatory mechanism that restores GnRH/LH pulse generation.

Kisspeptin, encoded by Kiss1, is essential for reproduction in mammals. Kiss1 expression is regulated by estrogen via histone acetylation in the Kiss1 promotor region. Thus, elucidation of histone modification factor(s) involved in the regulation of Kiss1 expression is required to gain further understanding of the mechanisms of its control. The RNA-seq analysis of isolated kisspeptin neurons, obtained from the arcuate nucleus (ARC) of female rats, revealed that Rbbp7, encoding retinoblastoma binding protein 7 (RBBP7), a member of histone modification and chromatin remodeling complexes, is highly expressed in the ARC kisspeptin neurons. Thus, the present study aimed to investigate whether RBBP7 is involved in Kiss1 expression. Histological analysis using in situ hybridization (ISH) revealed that Rbbp7 expression was located in several hypothalamic nuclei, including the ARC and the anteroventral periventricular nucleus (AVPV), where kisspeptin neurons are located. Double ISH for Rbbp7 and Kiss1 showed that a majority of kisspeptin neurons (more than 85%) expressed Rbbp7 mRNA in both the ARC and the AVPV of female rats. Further, Rbbp7 mRNA knockdown significantly decreased in vitro expression of Kiss1 in a mouse immortalized kisspeptin neuronal cell line (mHypoA-55). Estrogen treatment significantly decreased and increased Kiss1 mRNA levels in the ARC and AVPV of ovariectomized female rats, respectively, but failed to affect Rbbp7 mRNA levels in both the nuclei. Taken together, these findings suggest that RBBP7 is involved in the upregulation of Kiss1 expression in kisspeptin neurons of rodents in an estrogen-independent manner.

Kisspeptin has an indispensable role in gonadotropin-releasing hormone/gonadotropin secretion in mammals. In rodents, kisspeptin neurons are located in distinct brain regions, namely the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN), arcuate nucleus (ARC), and medial amygdala (MeA). Among them, the physiological role of AVPV/PeN kisspeptin neurons in males has not been clarified yet. The present study aims to investigate the acute effects of the olfactory and/or mating stimulus with a female rat on hypothalamic and MeA Kiss1 mRNA expression, plasma luteinizing hormone (LH) and testosterone levels in male rats. Intact male rats were exposed to the following stimuli: exposure to clean bedding; exposure to female-soiled bedding as a female-olfactory stimulus; exposure to female-soiled bedding and mating stimulus with a female rat. The mating stimulus significantly increased the number of the AVPV/PeN Kiss1 mRNA-expressing cells in males within 5 minutes after the exposure, and significantly increased LH and testosterone levels, followed by an increase in male sexual behavior. Whereas, the males exposed to female-soiled bedding showed a moderate increase in LH levels and no significant change in testosterone levels and the number of the AVPV/PeN Kiss1 mRNA-expressing cells. Importantly, none of the stimuli affected the number of Kiss1 mRNA-expressing cells in the ARC and MeA. These results suggest that the mating-induced increase in AVPV/PeN Kiss1 mRNA expression may be, at least partly, involved in stimulating LH and testosterone release, and might consequently ensure male mating behavior. This study would be the first report suggesting that the AVPV/PeN kisspeptin neurons in males may play a physiological role in ensuring male reproductive performance.

The present study aimed to evaluate whether novel conditional kisspeptin neuron-specific Kiss1 knockout (KO) mice utilizing the Cre-loxP system could recapitulate the infertility of global Kiss1 KO models, thereby providing further evidence for the fundamental role of hypothalamic kisspeptin neurons in regulating mammalian reproduction. We generated Kiss1-floxed mice and hypothalamic kisspeptin neuron-specific Cre-expressing transgenic mice and then crossed these two lines. The conditional Kiss1 KO mice showed pubertal failure along with a suppression of gonadotropin secretion and ovarian atrophy. These results indicate that newly-created hypothalamic Kiss1 KO mice obtained by the Cre-loxP system recapitulated the infertility of global Kiss1 KO models, suggesting that hypothalamic kisspeptin, but not peripheral kisspeptin, is critical for reproduction. Importantly, these Kiss1-floxed mice are now available and will be a valuable tool for detailed analyses of roles of each population of kisspeptin neurons in the brain and peripheral kisspeptin-producing cells by the spatiotemporal-specific manipulation of Cre expression.

Increasing evidence shows that puberty onset is largely dependent on body weight rather than chronological age. To investigate the mechanism involved in the energetic control of puberty onset, the present study examined effects of chronic food restriction during the prepubertal period and the resumption of ad libitum feeding for 24 and 48 h on estrous cyclicity, Kiss1 (kisspeptin gene), Tac3 (neurokinin B gene) and Pdyn (dynorphin A gene) expression in the hypothalamus, luteinizing hormone (LH) secretion and follicular development in female rats. When animals weighed 75 g, they were subjected to a restricted feeding to retard growth to 70-80 g by 49 days of age. Then, animals were subjected to ad libitum feeding or remained food-restricted. The growth-retarded rats did not show puberty onset associated with suppression of both Kiss1 and Pdyn expression in the arcuate nucleus (ARC). 24-h ad libitum feeding increased tonic LH secretion and the number of Graafian and non-Graafian tertiary follicles with an increase in the numbers of ARC Kiss1- and Pdyn-expressing cells. 48-h ad libitum feeding induced the vaginal proestrus and a surge-like LH increase with an increase in Kiss1-expressing cells in the anteroventral periventricular nucleus (AVPV). These results suggest that the negative energy balance causes pubertal failure with suppression of ARC Kiss1 and Pdyn expression and then subsequent gonadotropin secretion and ovarian function, while the positive energetic cues trigger puberty onset via an increase in ARC Kiss1 and Pdyn expression and thus gonadotropin secretion and follicular development in female rats.

Kisspeptin, identified as a natural ligand of GPR54 in 2001, is now considered as a master regulator of puberty and subsequent reproductive functions in mammals. Our previous studies using Kiss1 knockout (KO) rats clearly demonstrated the indispensable role of kisspeptin in gonadotropin-releasing hormone (GnRH)/gonadotropin secretion. In addition, behavioral analyses of Kiss1 KO rats revealed an organizational effect of kisspeptin on neural circuits controlling sexual behaviors. Our studies using transgenic mice carrying a region-specific Kiss1 enhancer-driven reporter gene provided a clue as to the mechanism by which estrogen regulates Kiss1 expression in hypothalamic kisspeptin neurons. Analyses of Kiss1 expression and gonadotropin secretion during the pubertal transition shed light on the mechanism triggering GnRH/gonadotropin secretion at the onset of puberty in rats. Here, we summarize data obtained from the aforementioned studies and revisit the physiological roles of kisspeptin in the mechanism underlying reproductive functions in mammals.

Puberty is associated with an increase in gonadotropin secretion as a result of an increase in gonadotropin-releasing hormone (GnRH) secretion. Kisspeptin is considered to play a key role in puberty onset in many mammalian species, including rodents, ruminants and primates. The present study aimed to determine if changes in hypothalamic expression of the KISS1 gene, encoding kisspeptin, are associated with the onset of puberty in pigs. The animals (n=4 in each group) were perfused with 4% paraformaldehyde at 0, 1, 2, 3 and 4 months old, as prepubertal stages, and at 5 months old, as the peripubertal stage, following each blood sampling. KISS1 gene expressions in coronal sections of brains were visualized by in situ hybridization. Plasma luteinizing hormone (LH) was measured by radioimmunoassay. KISS1 mRNA signals were observed in the arcuate nucleus (ARC) at all ages examined without any significant difference in the number of KISS1-expressing cells, indicating that the KISS1 gene is constantly expressed in the ARC throughout pubertal development in pigs. The plasma LH concentration was the highest in 0-month-old piglets and significantly decreased in the 1- and 2 month-old groups (P

The mammalian ovary is an extremely dynamic organ in which a large majority of follicles are effectively eliminated throughout their reproductive life. Due to the numerous efforts of researchers, mechanisms regulating follicular growth and atresia in mammalian ovaries have been clarified, not only their systemic regulation by hormones (gonadotropins) but also their intraovarian regulation by gonadal steroids, growth factors, cytokines and intracellular proteins. Granulosa cells in particular have been demonstrated to play a major role in deciding the fate of follicles, serving molecules that are essential for follicular growth and maintenance as well as killing themselves by an apoptotic process that results in follicular atresia. In this review, we discuss the factors that govern follicular growth and atresia, with a special focus on their regulation by granulosa cells. First, ovarian folliculogenesis in adult life is outlined. Then, we explain about the regulation of follicular growth and atresia by granulosa cells, in which hormones, growth factors and cytokines, death ligand-receptor system and B cell lymphoma/leukemia 2 (BCL2) family members (mitochondria-mediated apoptosis) are further discussed.

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice and plays an important role in other mammals including humans. Intraperitoneal (i.p.) injections with anti-LIF antibody (7.5 µg/g body weight, 3 times) between D3 (D1 = day of vaginal plug detection) and D4 effectively blocked embryo implantation; complete inhibition was achieved in C57BL/6J mice, and implantation was dramatically reduced in ICR mice (reduced to 27%). Normal rabbit IgG used as the control did not disturb embryo implantation. Anti-LIF antibody was localized not only in the stroma, but also in the luminal epithelium and the glandular lumen after i.p. injections. Growth-arrested blastocysts were recovered from the uterus without any implantation sites in both strains. Blastocysts made contact with the LE on the antimesometrial side; however, uterine stromal cells did not undergo secondary decidual reaction, and the uterine lumen was open, even at D7. Several regions of decidualization in ICR mice treated with anti-LIF antibody were smaller than those of the control, and development of blastocysts was delayed. The expression of LIF-regulated genes, such as immune-responsive gene-1 and insulin-like growth factor binding protein-3, was significantly decreased in C57BL/6J mice treated with anti-LIF antibody compared with the control, but not in ICR mice. The present study demonstrated that simple ip injections of an antibody are sufficient to block one of the important factors involved in embryo implantation in mice, and this method should also be easily applicable to the investigation of other factors involved in implantation.

In mammalian ovaries, most follicles are lost by atresia before ovulation. It has become apparent that the apoptosis of granulosa cells induces follicular atresia. Forkhead box O3 (FOXO3), also called FKHRL1 (forkhead in rhabdomyosarcoma-like 1), is a proapoptotic molecule that belongs to the FOXO subfamily of forkhead transcription factors. Foxo3-deficient female mice were reported to be infertile because of abnormal ovarian follicular development, but the precise influences of FOXO3 on follicular atresia of mature ovary have not been determined. Therefore, we examined the expression and function of FOXO3 in porcine ovarian follicles and granulosa-derived cells. FOXO3 mRNA levels in granulosa cells of porcine ovaries increased during atresia, while FOXO3 protein was abundant in granulosa cells of early atretic follicles. By immunohistochemistry, the inner surface area of the granulosa layer in early atretic follicles was strongly stained with anti-FOXO3 antibody. The granulosa cells expressing FOXO3 coincided with apoptotic cells, indicating a role of FOXO3 as a proapoptotic factor in granulosa cells of porcine ovaries. In porcine (JC-410) and human (KGN) granulosa-derived cells, cell death was induced by transfection of FOXO3 expression vectors. Expression of the proapoptotic factors Fas ligand (FASLG) and BCL2-like 11 (BCL2L11) was upregulated by FOXO3 in KGN cells. In conclusion, FOXO3 is expressed in porcine ovarian follicles and induces apoptosis in granulosa cells, suggesting that it is a candidate for the initiator of follicular atresia.

More than 99% of follicles in mammalian ovaries undergo atresia, but the mechanisms regulating the strict selection process are still unclear. Granulosa cell apoptosis is considered the trigger of follicular atresia, which occurs in advance of the death of an oocyte. Cellular FLICE-like inhibitory protein (cFLIP), a homologue of procaspase-8 (also called FLICE), is an intracellular anti-apoptotic protein. It is expressed in granulosa cells of porcine ovaries, where its levels decreases during follicular atresia. We hypothesized that cFLIP regulates granulosa cell apoptosis by acting as a pro-survival factor. In the present study, to further reveal the function of cFLIP in granulosa cells, we examined the anti-apoptotic mechanism of cFLIP using KGN, a human granulosa tumor cell line. Fas-mediated apoptosis was induced by co-treatment with anti-Fas antibody (CH-11), which acts as an agonist of Fas-ligand, and cycloheximide (CHX). When cFLIP was stably expressed in KGN cells following transfection of an expression vector, the Fas-mediated apoptosis was inhibited. Suppression of cFLIP by small interfering RNA (siRNA) spontaneously induced cell death. Silencing of cFLIP promoted cleavage of procaspase-8, and the cell death caused by cFLIP siRNA was completely blocked by a caspase-8 inhibitor (Z-IETD-FMK), indicating that cFLIP regulates apoptosis in KGN cells by inhibiting cleavage of procaspase-8. In conclusion, cFLIP is an essential pro-survival factor for granulosa cells, and it prevents granulosa cell apoptosis by inhibiting procaspase-8 activation.

The apoptosis inhibitory ligand (Netrin-1) and its receptor (p53-regulated receptor for death and life: p53RDL1) play an important role in the regulation of selective apoptosis. When Netrin-1 binds to p53RDL1, p53-dependent apoptosis is inhibited. We identified porcine (Sus scrofa) cDNAs encoding Netrin-1 [pNetrin-1; 1,803 base pairs (bp) and 600 amino acids (aa)] and p53RDL1 (pp53RDL1; 2,838 bp and 945 aa). Porcine p53RDL1 (pp53RDL1) contains a death domain (DD), a tandem specific amino acid region, in its C-terminal, suggesting that it mediates death signaling by binding with other pro-apoptotic factors via the DD. Porcine Netrin-1 (pNetrin-1), pp53RDL1 and the DD in pp53RDL1 showed high levels of identity in aa sequence with human and murine Netrin-1 (98 and 97%, respectively), p53RDL1 (94 and 91%, respectively) and the DD in p53RDL1 (96 and 95%, respectively). Reverse transcription-polymerase chain reaction (RT-PCR) revealed that the levels of pNetrin-1 and pp53RDL1 mRNAs were moderate in granulosa cells compared with their expression in other tissues and that their levels during follicular atresia were stable. The Netrin-1 and p53RDL1 system may regulate the induction of apoptosis in porcine granulosa cells.

More than 99% of follicles undergo a degenerative process known as "atresia" in mammalian ovaries, and only a few follicles ovulate during follicular growth and development. Follicular selection predominantly depends on granulosa cell apoptosis. To reveal the molecular mechanisms of selective follicular atresia, we examined the changes in the levels of interleukin-6 (IL-6) receptors expressed in the granulosa cells of pig ovaries. The levels of IL-6 receptor (IL-6R)-alpha mRNA and protein in granulosa cells were quantified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. IL-6R alpha mRNA and protein were highly expressed in the granulosa cells of progressed atretic follicles. Enzyme-linked immunosorbent assay showed that the expression of IL-6 soluble receptor (IL-6sR) protein in follicular fluid decreased during atresia. Moreover, we isolated porcine cDNA encoding an IL-6 signal transducer, gp130. Porcine gp130 (2,754 bp and 917 amino acids) was identified from a cDNA library prepared using follicular granulosa cells of pig ovaries. Porcine gp130 was highly homologous with human and murine gp130. RT-PCR analysis revealed that the level of gp130 mRNA also decreased during atresia. We presume that IL-6sR and gp130, but not IL-6R alpha, play important roles in regulation of granulosa cell survival.

To reveal the mechanisms regulating the selective atresia of follicles in porcine ovaries, we examined the changes in the mRNA and protein levels of cell-death ligand, Fas/APO-1/CD95 ligand (FasL), and its receptor, Fas/APO-1/CD95 (Fas), and the localization of the proteins in granulosa cells during follicular atresia using the reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical techniques, respectively. Trace levels of FasL mRNA and protein were detected in the granulosa cells of healthy follicles; however, weak levels were detected in those of early atretic follicles, and the levels increased during atresia. Trace/weak levels of Fas mRNA and protein were detected in the granulosa cells of healthy follicles. Fas protein was located in the cytoplasmic area, not in cell membrane area, indicating that it has no activity in regard to inducing apoptosis. When apoptosis commences in granulosa cells, Fas moves from the cytoplasmic to cell membrane area. FasL and Fas mRNAs and proteins in granulosa cells were upregulated during follicular atresia. The FasL and Fas system may play a crucial role in the regulation of apoptosis in granulosa cells during selective follicular atresia in porcine ovaries.

To reveal the specific regulatory molecules that control granulosa cell apoptosis during follicular atresia, we immunohistochemically examined the localization of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors in porcine ovaries. A marked reduction in the expression of decoy receptor-1 (DcR1), which has high affinity for TRAIL, was demonstrated in granulosa cells of atretic follicles, but no marked differences were seen in expression of TRAIL or other TRAIL-receptors (death receptor-4 or death receptor-5) in granulosa cells between healthy and atretic follicles. No positive staining for DcR2 was seen. We presum that TRAIL and its receptors are involved in induction of apoptosis in granulosa cells during atresia, and that DcR1 plays an inhibitory role in granulosa cell apoptosis.

Previously, we histochemically demonstrated the expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors, death receptor-4 (DR4), death receptor-5 (DR5) and decoy receptor-1 (DcR1) in granulosa cells of porcine follicles. However, TRAIL can induce both cell death and cell proliferation. In the present study, reverse transcription polymerase chain reaction and in situ hybridization analyses revealed increased mRNA expression of TNF receptor-associated death domain protein (TRADD), which transmits the death signal from DR4 and/or DR5 to intracellular apoptosis-signal transduction components, in granulosa cells was demonstrated only in atretic follicles but not in healthy follicles. These findings indicate that TRADD is involved in induction of apoptosis in granulosa cells, and that the TRAIL-receptor system induces apoptosis in granulosa cells during atresia in porcine ovaries.

Determination of the expression level and localization of cell adhesion molecules is crucial for understanding the mechanism of maintenance and remodeling of the ovarian follicle structure. We immunocytochemically investigated expression of the cell adhesion molecules, "classic" cadherins and β-catenin, in developing and/or atretic follicles of porcine ovaries. Healthy follicles showed strong staining for cadherin-8 and β-catenin in granulosa cells tightly attached to the basement membrane, and moderate/weak staining was seen on the inner surface of the granulosa cell layer. Strong VE-cadherin expression was seen in a single cell layer attached to the basement membrane in the theca interna layer of healthy follicles. The expression of cadherin-8, β-catenin and VE-cadherin decreased during follicular atresia. No positive staining was observed for R-cadherin, E-cadherin, T-cadherin, BR-cadherin or P-cadherin, and a weak positive reaction for N-cadherin was seen only in the granulosa cells of healthy and early atretic follicles. These findings indicate that cadherin-8, N-cadherin and VE-cadherin have important roles in follicular development and/or degeneration, and that decreases in the expression of these cadherins are involved in follicular atresia in porcine follicles.