Assessing recrudescence of photoregressed Siberian hamster ovaries using in vitro whole ovary culture.

In vitro culture has been used to study different aspects of ovarian function; however, this technique has not been applied to study recrudescence, or the return of ovarian function in seasonally breeding species. In Siberian hamsters, exposure to inhibitory photoperiods induces declines in ovarian function, which are restored with photostimulation. Because these changes are mediated by changes in systemic gonadotropin (GT) secretion, we hypothesized that culturing photoregressed ovaries with GT would restore aspects of function and induce expression of key folliculogenic factors. Adult female Siberian hamsters were exposed to either long-day (LD; 16L:8D) or short-day (SD; 8L:16D) photoperiods for 14 weeks to maintain in vivo cyclicity or induce gonadal regression, respectively. Isolated ovaries were then cultured for 10 days with or without GT. Ovarian mass and messenger RNA (mRNA) expression of mitotic marker Pcna were increased in cultured SD ovaries (cSD) ovaries with GT as compared to without GT, with no changes noted among cultured LD (cLD) ovaries. Media estradiol and progesterone concentrations increased in both cLD and cSD ovaries cultured with GT as compared to without GT. No differences in follicle numbers or incidence of apoptosis were noted across groups. In addition, differential mRNA expression of folliculogenic growth factors ( Bmp-4, Ntf-3, Inh-α, Gdf-9, Igf-1, Has-2, and Cox-2) was observed in cSD treated with or without GT. Together, these results suggest that this in vitro model could be a useful tool to (a) study the return of function in photoregressed ovaries, and (b) to identify the specific roles folliculogenic factors play in ovarian recrudescence.

Matrix metalloproteinase inhibition influences aspects of photoperiod stimulated ovarian recrudescence in Siberian hamsters.

Blocking matrix metalloproteinase (MMP) activity in vivo with inhibitor GM6001 impedes photostimulated ovarian recrudescence in photoregressed Siberian hamsters. Since direct and indirect effects of MMPs influence a myriad of ovarian functions, we investigated the effect of in vivo MMP inhibition during recrudescence on ovarian mRNA expression of steroidogenic acute regulatory protein (StAR), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), Cyp19a1 aromatase, epidermal growth factor receptor (EGFR), amphiregulin (Areg), estrogen receptors (Esr1 and Esr2), tissue inhibitors of MMPs (TIMP-1,-2,-3), proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor A (VEGFA), its receptor VEGFR-2, and angiopoietin-2 (Ang-2). Female Siberian hamsters were randomly assigned to one of four photoperiod groups: stimulatory long (LD) or inhibitory short (SD) photoperiods, or transferred from SD to LD for 2 weeks (post-transfer, PT). Half of the PT hamsters were injected (ip) daily with GM6001 (PTG). SD exposure reduced ovarian StAR, 3ß-HSD, Cyp19a1, Esr1, Esr2, TIMPs 2-3, PCNA, VEGFR-2 and Ang-2 mRNA expression (p<0.05), and 2 weeks of photostimulation restored mRNA expression of 3ß-HSD and PCNA and increased Areg and VEGFA mRNA expression in the PT group. GM6001 treatment during photostimulation (PTG) increased TIMP-1, -2 and -3 and PCNA mRNA, but inhibited Areg mRNA expression compared to PT. Neither photoperiod nor GM6001 altered EGFR expression. Results of this study suggest that in vivo inhibition of MMP activity by GM6001 may impede ovarian recrudescence, particularly follicular growth, in two ways: (1) directly by partially inhibiting the release of EGFR ligands like Areg, thereby potentially affecting EGFR activation and its downstream pathway, and (2) indirectly by its effect on TIMPs which themselves can affect proliferation, angiogenesis and follicular growth.

Anti-Müllerian hormone (AMH), inhibin-α, growth differentiation factor 9 (GDF9), and bone morphogenic protein-15 (BMP15) mRNA and protein are influenced by photoperiod-induced ovarian regression and recrudescence in Siberian hamster ovaries.

Exposure of Siberian hamsters to short photoperiod (SD) inhibits ovarian function, including folliculogenesis, whereas function is restored with their transfer to long photoperiods (LD). To investigate the mechanism of photo-stimulated recrudescence, we assessed key folliculogenic factors-anti-Müllerian hormone (AMH), inhibin-α, growth differentiation factor-9 (GDF9), and bone morphogenic protein-15 (BMP15)-across the estrus cycle and in photo-regressed and recrudescing ovaries. Adult hamsters were exposed to either LD or SD for 14 weeks, which respectively represent functional and regressed ovaries. Select regressed hamsters were transferred back to LD for 2 (post-transfer week 2; PTw2) or 8 weeks (PTw8). Ovaries were collected and fixed in formalin for immunohistochemistry or frozen in liquid nitrogen for real-time PCR. AMH, inhibin-α, GDF9, and BMP15 mRNA and protein were detected in all stages of the estrus cycle. Fourteen weeks of SD exposure increased (P < 0.05) ovarian AMH, GDF9, and BMP15, but not inhibin-α mRNA levels as compared to LD. Transfer of regressed hamsters to stimulatory long photoperiod for 8 weeks returned AMH and GDF9 mRNA levels to LD-treated levels, and further increased mRNA levels for inhibin-α and BMP15. Immunostaining for AMH, inhibin-α, GDF9, and BMP15 proteins was most intense in preantral/antral follicles and oocytes. The overall immunostaining extent for AMH and inhibin-α generally mirrored the mRNA data, though no changes were observed for GDF9 or BMP15 immunostaining. Shifts in mRNA and protein levels across photoperiod conditions suggest possible syncretic roles for these folliculogenic factors in photo-stimulated recrudescence via potential regulation of follicle recruitment, preservation, and development.

Intraovarian expression of GnRH-1 and gonadotropin mRNA and protein levels in Siberian hamsters during the estrus cycle and photoperiod induced regression/recrudescence.

The hypothalamic-pituitary-gonadal (HPG) axis is the key reproductive regulator in vertebrates. While gonadotropin releasing hormone (GnRH), follicle stimulating (FSH), and luteinizing (LH) hormones are primarily produced in the hypothalamus and pituitary, they can be synthesized in the gonads, suggesting an intraovarian GnRH-gonadotropin axis. Because these hormones are critical for follicle maturation and steroidogenesis, we hypothesized that this intraovarian axis may be important in photoperiod-induced ovarian regression/recrudescence in seasonal breeders. Thus, we investigated GnRH-1 and gonadotropin mRNA and protein expression in Siberian hamster ovaries during (1) the estrous cycle; where ovaries from cycling long day hamsters (LD;16L:8D) were collected at proestrus, estrus, diestrus I, and diestrus II and (2) during photoperiod induced regression/recrudescence; where ovaries were collected from hamsters exposed to 14 weeks of LD, short days (SD;8L:16D), or 8 weeks post-transfer to LD after 14 weeks SD (PT). GnRH-1, LHß, FSHß, and common α subunit mRNA expression was observed in cycling ovaries. GnRH-1 expression peaked at diestrus I compared to other stages (p < 0.05). FSHß and LHß mRNA levels peaked at proestrus and diestrus I (p < 0.05), with no change in the α subunit across the cycle (p > 0.05). SD exposure decreased ovarian mass and plasma estradiol concentrations (p<0.05) and increased GnRH-1, LHß, FSHß, and α subunit mRNA expression as compared to LD and, except for LH, compared to PT (p < 0.05). GnRH and gonadotropin protein was also dynamically expressed across the estrous cycle and photoperiod exposure. The presence of cycling intraovarian GnRH-1 and gonadotropin mRNA suggests that these hormones may be locally involved in ovarian maintenance during SD regression and/or could potentially serve to prime ovaries for rapid recrudescence.

Inhibition of matrix metalloproteinases in Siberian hamsters impedes photostimulated recrudescence of ovaries.

Exposure of Siberian hamsters to short photoperiod for 14 weeks induces ovarian regression. Subsequent transfer to long photoperiod restores ovarian function, and 2 weeks of photostimulation increases plasma estradiol (E(2)), antral follicles, and corpora lutea (CL). Because tissue remodeling involved with photostimulated ovarian recrudescence is associated with differential expression of matrix metalloproteinases (MMPs), we hypothesized that inhibiting MMP activity using a broad-spectrum in vivo MMP inhibitor, GM6001, would curtail recrudescence. One group of hamsters was placed in long days (LD; 16 h light:8 h darkness) for 16 weeks. Another group was placed in inhibitory short days (SD; 8 h light:16 h darkness) for 14 weeks. A third group was placed in SD for 14 weeks and transferred to LD for 2 weeks to stimulate recrudescence. During weeks 14-16, animals were either not treated or treated daily with i.p. injections of GM6001 (20 mg/kg) or vehicle (DMSO). GM6001 reduced gelatinase activity and decreased immunohistochemical staining for MMP1, MMP2, and MMP3 compared with vehicle. No differences between controls, vehicle, or GM6001 treatment were observed among LD animals, despite a trend toward reduction in CL and E(2) with GM6001. Although SD reduced ovarian function, photostimulation of transferred controls increased uterine mass, plasma E(2), appearance of antral follicles, and CL. With GM6001 treatment, photostimulation failed to increase uterine mass, plasma E(2), antral follicles, or CL. These data show, for the first time, that in vivo GM6001 administration inhibits MMP activity in hamster ovaries during photostimulation, and indicate that this inhibition may impede photostimulated recrudescence of ovaries. This study suggests an intriguing link between MMP activity and return to ovarian function during photostimulated recrudescence.

Differential ovarian expression of KiSS-1 and GPR-54 during the estrous cycle and photoperiod induced recrudescence in Siberian hamsters (Phodopus sungorus).

Kisspeptins, coded by the KiSS-1 gene, regulate aspects of the reproductive axis by stimulating GnRH release via the G protein coupled receptor, GPR54. Recent reports show that KiSS/GPR54 may be key mediators in photoperiod-controlled reproduction in seasonal breeders, and that KiSS-1/GPR54 are expressed in the hypothalamus, ovaries, placenta, and pancreas. This study examined the expression of KiSS-1/GPR54 mRNA and protein in ovaries of Siberian hamsters (Phodopus sungorus). Ovaries from cycling hamsters were collected during proestrus (P), estrus (E), diestrus I (DI), and diestrus II (DII). To examine KiSS-1/GPR54 during stimulated recrudescence, additional hamsters were maintained either in long day (LD 16L:8D, control) or short day (SD 8L:16D) for 14 weeks and then transferred to LD for 0-8 weeks. Staining of KiSS-1/GPR54 protein was detected by immunohistochemistry in steroidogenic cells of pre-antral and antral follicles, and corpora lutea. Immunostaining peaked in P and E, but decreased in the diestrus stages (P < 0.05). In recrudescing ovaries, KiSS-1/GPR54 immunostaining was low after 14 weeks of SD exposure (post-transfer [PT] week 0), and increased during the early weeks of recrudescence. Expression of KiSS-1/GPR54 mRNA was low with short day exposure, but increased during recrudescence and was higher at PT week 8 as compared to PT weeks 0 and 2 (P < 0.05). The elevated KiSS-1/GPR54 expression during P and E suggests a potential role in ovulation in Siberian hamsters. Transient increases in KiSS-1/GPR54 expression following LD stimulation are also suggestive of possible involvement in ovulation and/or restoration of ovarian function.

Differential activity of matrix metalloproteinases (MMPs) during photoperiod induced uterine regression and recrudescence in Siberian hamsters (Phodopus sungorus).

Siberian hamsters adapt to seasonal changes by reducing their reproductive function during short days (SD). SD exposure reduces uterine mass and reproductive capacity, but underlying cellular mechanisms remain unknown. Because matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in uterine development, parturition, and postpartum remodeling, their expression in uterine tissue from Siberian hamsters undergoing photoperiod-mediated reproductive regression and recrudescence was investigated. Female hamsters were exposed to long day (LD, 16L:8D, controls) or SD (8L:16D) for 3-12 weeks (regression); a second group was exposed to SD or LD for 14 weeks and then transferred to LD for 0-8 weeks (recrudescence). Hamsters were euthanized, uteri collected, and homogenates analyzed by gelatin zymography or Western blotting for MMP and TIMP protein levels. Uterine weight decreased (67-75%) at SD weeks 12-14 and increased post-LD transfer (PT) reaching LD values by PT week 2. MMP-2, but not MMP-9 activity was reduced by SD week 12 or 14 but increased to LD levels at PT week 2. MMP-3 expression increased at SD week 9 compared to other SD and LD groups. MMP-14 and -13 protein levels decreased at SD week 3 but returned to LD levels by SD week 6. During recrudescence, MMP-3 (PT weeks 0-2), MMP-13 (PT week 4), and MMP-14 (PT weeks 2, 4) protein levels were higher than LD. TIMP-1 and 2 were present at low levels. Significant and differential variations in uterine MMP activity/expression during photoperiod-induced regression and recrudescence were observed. These changes likely reflect increases in tissue remodeling during both the adaptation to SD and the restoration of reproductive function.

Rapid changes in ovarian mRNA induced by brief photostimulation in Siberian hamsters (Phodopus sungorus).

This study sought to characterize the rapid intraovarian mRNA response of key folliculogenic factors that may contribute to the restoration of folliculogenesis during 2-10 days of photostimulation in Siberian hamsters. Adult hamsters were exposed to short photoperiod (8L:16D) for 14 weeks (SD). A subset were then transferred to long photoperiod (16L:8D) for 2 (PT day-2), 4 (PT day-4), or 10 days (PT day-10). Quantitative real-time PCR was used to measure intraovarian mRNA expression of: gonadotropin releasing hormone (GnRH), follicle stimulating hormone ß-subunit (FSHß-subunit), luteinizing hormone ß-subunit (LHß-subunit), FSH and LH receptors, estrogen receptors α and ß (Esr1 and Esr2), matrix metalloproteinase (MMP)-2 and -9, anti-Müllerian hormone (AMH), inhibin-α subunit, fibroblast growth factor-2 (FGF-2) and proliferating cell nuclear antigen (PCNA). Compared to SD, plasma FSH concentrations increased on PT day-4 and the number of antral follicles and corpora lutea increased on PT day-10. FSHR and inhibin-α mRNA expression also increased on PT day-4, whereas LHR and proliferation marker PCNA both increased on PT day-10 as compared to SD. Esr1 mRNA increased on PT day-2 and remained significantly increased as compared to SD, whereas Esr1 mRNA increased only on PT day-2, similar to FGF-2 and MMP-2 results. No differences were observed in mRNA expression in ovarian GnRH, FSHß- and LHß-subunits, AMH, and MMP-9 mRNA with 2-10 days of photostimulation. Rapid increases in intraovarian FSHR and inhibin-α mRNA and antral follicle/corpora lutea numbers suggest that the ovary is primed to react quickly to the FSH released in response to brief periods of photostimulation.

Differential expression of matrix metalloproteinases during stimulated ovarian recrudescence in Siberian hamsters (Phodopus sungorus).

The matrix metalloproteinases (MMPs) are a family of extracellular matrix-cleaving enzymes involved in ovarian remodeling. In many non-tropical species, including Siberian hamsters, ovarian remodeling is necessary for the functional changes associated with seasonal reproduction. We evaluated MMPs and their endogenous inhibitors (TIMPs), during photoperiod-induced ovarian recrudescence in Siberian hamsters. Hamsters were transferred from long day (LD; 16:8) to short day (SD; 8:16) photoperiods for 14weeks, and then returned to LD for 0, 1, 2, 4, or 8weeks for collection of ovaries and plasma. Post-transfer (PT) LD exposure increased body and ovarian mass. Number of corpora lutea and antral, but not preantral follicles increased in PT groups. Plasma estradiol concentrations were lower in PT weeks 0-4, and returned to LD levels at PT week 8. No change was observed in relative MMP/TIMP mRNA levels at PT week 0 (SD week 14) as compared to LD. Photostimulation increased MMP-2 mRNA at PT week 8 as compared to PT weeks 0-1. MMP-14 mRNA expression peaked at PT weeks 1-2 as compared to LD levels, while MMP-13 expression was low during this time. TIMP-1 mRNA peaked at PT week 8 as compared to PT weeks 0-4. No changes were noted in MMP-9 and TIMP-2 mRNA expression. In general, MMP/TIMP protein immunodetection followed the same patterns with most staining occurring in granulosa cells of follicles and corpora lutea. Our data suggest that mRNA and protein for several members of the MMP/TIMP families are expressed in Siberian hamster ovaries during recrudescence. Because of the variation observed in expression patterns, MMPs and TIMPs may be differentially involved with photostimulated return to ovarian function.

Interaction of MAPK and 12-lipoxygenase pathways in growth and matrix protein expression in mesangial cells.

The lipoxygenase (LO) pathway of arachidonate metabolism and mitogen-activated protein kinases (MAPKs) can mediate cellular growth and ANG II effects in vascular smooth muscle cells. However, their role in renal mesangial cells (MC) is not very clear. ANG II treatment of rat MC significantly increased 12-LO mRNA expression and formation of the 12-LO product 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE; P < 0.03]. ANG II-induced [(3)H]leucine incorporation was blocked by an LO inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (P < 0.02). 12(S)-HETE and ANG II directly induced cellular hypertrophy and fibronectin (FN) expression (P < 0.01) to a similar extent. ANG II and 12(S)-HETE led to activation of p38(MAPK) and its target transcription factor cAMP-responsive element-binding protein (CREB). ANG II- and 12(S)-HETE-induced CREB activation and [(3)H]leucine incorporation were blocked by the p38(MAPK) inhibitor SB-202190. A specific molecular inhibitor of rat 12-LO mRNA, namely, a novel ribozyme, could attenuate ANG II-induced FN mRNA. Thus p38(MAPK)-dependent CREB activation may mediate ANG II- and LO product-induced FN expression and cellular growth in rat MC. ANG II effects may be mediated by the LO pathway. These results suggest a novel interaction between LO and p38(MAPK) activation in MC matrix synthesis associated with renal complications.

Role of 12-lipoxygenase in the stimulation of p38 mitogen-activated protein kinase and collagen alpha5(IV) in experimental diabetic nephropathy and in glucose-stimulated podocytes.

The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism is implicated in extracellular matrix (ECM) synthesis, but its role in podocytes has not been studied. This study tested whether 12-LO induction by diabetes or by high glucose (HG) in cultured podocytes alters glomerular basement membrane by activating signal transduction pathways culminating in ECM synthesis. Sprague-Dawley rats received an injection of diluent (control [C]) or streptozotocin 65 mg/kg (DM) and were killed at 1 or 4 mo. Glomerular 12-LO mRNA and protein levels were higher in DM than in C glomeruli at 1 and 4 mo, and 12-LO localized predominantly in podocytes. Glomerular p38 mRNA and protein were higher in DM at months 1 and 4, but phospho-p38 mitogen-activated protein (MAPK) was increased only at month 1. Glomerular collagen alpha5(IV)/glutaraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratio was increased in DM at month 1 but not at month 4, whereas collagen alpha5(IV) protein was higher at both 1 and 4 mo. Mouse podocytes were cultured in media with 25 mM glucose (HG) with or without the 12-LO inhibitor cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC) or with 5.5 mM glucose + 19.5 mM mannitol (low glucose [LG+M]) for 10 d at 37 degrees C. 12-LO mRNA and protein levels were higher in HG than in LG+M as was the p38 MAPK/GAPDH mRNA ratio. Phospho-p38 MAPK protein but not total p38 MAPK was higher in HG compared with LG+M. Collagen alpha5(IV)/GAPDH mRNA ratio and protein were higher in HG than in LG+M. 12-LO inhibition by CDC decreased HG-induced phospho-p38 MAPK and the phospho-p38/total p38 MAPK ratio, collagen alpha5(IV)/GAPDH mRNA ratio, and collagen alpha5(IV) protein expression. In summary, diabetes in vivo and exposure of podocytes to HG in vitro stimulated 12-LO, p38 MAPK, and collagen alpha5(IV) mRNA and (activated) protein. 12-LO inhibition by CDC diminished the expression of podocyte phospho-p38 MAPK and collagen alpha5(IV) mRNA and protein. These findings implicate 12-LO and the p38 MAPK signaling pathway in the mediation of ECM synthesis by podocytes in diabetes.