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Skeletal health consequences associated with inflammatory diseases of the airways significantly contribute to morbidity. Sex differences have been described independently for lung and bone diseases. Repetitive inhalant exposure to lipopolysaccharide (LPS) induces bone loss and deterioration in male mice, but comparison effects in females are unknown. Using an intranasal inhalation exposure model, 8-week-old C57BL/6 male and female mice were treated daily with LPS (100 ng) or saline for 3 weeks. Bronchoalveolar lavage fluids, lung tissues, tibias, bone marrow cells, and blood were collected. LPS-induced airway neutrophil influx, interleukin (IL)-6 and neutrophil chemoattractant levels, and bronchiolar inflammation were exaggerated in male animals as compared to female mice. Trabecular bone micro-CT imaging and analysis of the proximal tibia were conducted. Inhalant LPS exposures lead to deterioration of bone quality only in male mice (not females) marked by decreased bone mineral density, bone volume/tissue volume ratio, trabecular thickness and number, and increased bone surface-to-bone volume ratio. Serum pentraxin-2 levels were modulated by sex differences and LPS exposure. In proof-of-concept studies, ovarectomized female mice demonstrated LPS-induced bone deterioration, and estradiol supplementation of ovarectomized female mice and control male mice protected against LPS-induced bone deterioration findings. Collectively, sex-specific differences exist in LPS-induced airway inflammatory consequences with significant differences found in bone quantity and quality parameters. Male mice demonstrated susceptibility to bone loss and female animals were protected, which was modulated by estrogen. Therefore, sex differences influence the biologic response in the lung-bone inflammatory axis in response to inhalant LPS exposures.

Protocol for mass propagation of Bambusa bamboos (L.) Voss was developed through in vitro culture. Nodal segments containing pre-existing axillary bud, after surface sterilization, were inoculated on liquid Murashige and Skoog’s (MS) basal medium containing different concentrations and combinations of cytokinins (BAP, TDZ and Kn). The highest direct shoot induction (90%) was obtained in the MS liquid medium supplemented with 2.0 mg/l BAP and 1.0 mg/l TDZ with maximum average number of shoots (3.14 ± 0.06) per explant. Highest shoot multiplication (16.58 ± 0.24 shoots per culture) with highest average shoot length (9.21 ± 0.13 cm) was obtained when in vitro raised shoots were cultured in gelrite gelled MS medium in conjunction with 2.0 mg/l BAP and 1.0 mg/l TDZ. Incorporation of 10% coconut water with 4% sucrose in the above mentioned medium resulted satisfactory shoot growth and development with an average 26.7 ± 0.60 shoots per culture. For root induction, in vitro raised shoots were divided into clumps of 4-5 shoots in each clump and transferred onto both liquid and gelled half-strength MS medium containing different concentrations and combinations of auxins (IBA and NAA). Maximum rooting (86.67%) was achieved in half-strength of MS medium fortified with 2.5 mg/l IBA and 2.5 mg/l NAA with an average 8.72 ± 0.42 root per shoot. The rooted plantlets were then transferred to polybags containing garden soil, sand and compost mixture with 1:1:1 ratio. After a month the hardened plantlets were then transferred to the larger pots containing garden soil and compost with 1:1 ratio for sufficient growth and finally transplanted to the field. In this process, the highest 100% survivability was recorded from well-established rooted plantlets. The regenerated plants showed well developed root and shoot systems in field condition.Jahangirnagar University J. Biol. Sci. 5(2): 15-26, 2016 (December)

The usefulness of azaline B, a GnRH antagonist, in suppressing gonadotropin secretion in the golden hamster was examined by examining follicular development, steroidogenesis and expression of steroidogenic enzymes. Serum levels of P and E declined significantly, while FSH or LH was undetectable in azaline B-treated hamsters. FSH significantly increased serum E levels, whereas LH upregulated serum P levels. The formation of antral follicles ceased in azaline-treated hamsters, but was reversed by FSH with or without LH supplement. FSH also activated the primordial follicle pool resulting in increased formation of primary and preantral follicles. Further, an increasing trend in the formation of preantral follicles in response to E or E + P, and the formation of antral follicles in response to E + P treatment was evident. The level of Cyp11a1 mRNA increased markedly in LH- or LH + FSH-treated hamsters, whereas FSH with or without LH upregulated Cyp17a1, Cyp19a1 and Fshr mRNA expression. E without or with P also upregulated ovarian Cyp19a1 mRNA expression. The expression of enzyme protein corroborated the mRNA data. In summary, azaline B is an efficient GnRH antagonist in the hamster, and will be useful in studying the selective effect of gonadotropins on ovarian functions without disrupting the physiological functions of other hormones in ovarian cells.

Primordial follicle (PF) pool determines the availability of follicles for ovulation in all mammals. Premature depletion of the PF reserve leads to subfertility or infertility. Bone morphogenetic protein 2 (BMP2) promotes PF formation by facilitating oocyte and granulosa cell development. Estradiol-17β (E2) upregulates PF formation in developing hamster ovaries. However, if BMP2 mediates E2 effect is not known. We hypothesize that E2 facilitates the effect of BMP2 on somatic to granulosa cell transition. BMP2 and E2 together significantly upregulated the percentage of PFs in hamster fetal ovaries in vitro compared with either of the treatments alone. E2 also promoted BMP2 expression in vivo. Inhibition of BMP2 receptors suppressed E2-stimulation of PF formation while knockdown of BMP2 in vitro significantly suppressed the E2 effect. In contrast, estrogen receptor blocker did not affect BMP2 action. Inhibition of the activity of E2 or BMP2 receptors, either alone or combined during the last two days of the culture (C6-C8) resulted in a significant decrease in PF formation by C8, suggesting that both BMP2 and E2 action is essential for somatic cell differentiation for PF formation. Together, the results suggest that E2 activates BMP2-BMPR system leading to the formation of primordial follicles.

A large number of shoots regenerated from lateral buds and young leaves of Carica papaya L. cv. Shahi on MS supplemented with 1.0 mg/l zeatin and 0.2 mg/l NAA. Addition of 200 mg/l casein hydrolysate (CH) to the medium increased the number of shoots per culture and incorporation of 2.0 g/l activated charcoal (AC) to the medium resulted effective shoot growth with healthy leaf. While addition of 100 mg/l urea and 2.0 g/l activated charcoal to the medium showed proper shoot elongation. Best rooting was obtained from shoots cultured on half-strength of MS fortified with 4.0 mg/l IBA.Within four weeks of transfer to the rooting medium, 90% microcuttings produced 12 - 14 roots. The regenerated plantlets were successfully transferred to potted soil. About 84% plantlets survived in the experimental field. DOI: http://dx.doi.org/10.3329/bjb.v41i2.13448 Bangladesh J. Bot. 41(2): 191-195, 2012 (December)

An efficient protocol was developed for in vitro mass propagation of an important medicinal shrub, Plumbago zeylanica L., (Plumbaginaceae) through direct organogenesis using shoot tip and nodal explants. Best shoot induction was observed on MS basal medium supplemented with 0.5 mg/l BAP, in which 86.4% of nodal explants responded to produce maximum number (12.4 ± 0.66) of shoots per culture. In vitro raised shoots rooted on half strength MS medium with 0.5 mg/l IAA. For acclimatization and transplantation, the plantlets in the rooted culture tubes were kept in normal room temperature for 7 days before transplanting in pots where plantlets were reared for three weeks. The survival rate of regenerated plantlets was 85%. DOI: http://dx.doi.org/10.3329/bjsir.v47i3.13063 Bangladesh J. Sci. Ind. Res. 47(3), 297-302 2012

A protocol was established for in vitro shoot proliferation and plant regeneration of a rare medicinal shrub of Bangladesh, Plumbago indica L. (Plumbaginaceae) using shoot tip and nodal explants. Best shoot induction was observed on MS medium supplemented with 0.5 mg/l BAP, in which 92% of nodal explants responded to produce maximum number (42.8 ± 1.18) of shoots per explants. In vitro raised shoots rooted on half strength of MS medium with 0.5 mg/l IAA. For acclimatization and transplantation, the plantlets in the rooting culture tubes were kept in normal room temperature for 7 days before transplanting them in pots containing soil where plantlets were reared for three weeks. The survival rate of regenerated plantlets was 82%. DOI: http://dx.doi.org/10.3329/bjsir.v47i2.11452 Bangladesh J. Sci. Ind. Res. 47(2), 197-202, 2012

We assessed neonatal diethylstilbestrol (DES)-induced disruption at various endocrine levels in the hamster. In particular, we used organ transplantation into the hamster cheek pouch to determine whether abnormalities observed in the post-pubertal ovary are due to: (a) a direct (early) mechanism or (b) an indirect (late) mechanism that involves altered development and function of the hypothalamus and/or pituitary. Of the various disruption endpoints and attributes assessed: (1) some were consistent with the direct mechanism (altered uterine and cervical dimensions/organization, ovarian polyovular follicles, vaginal hypospadius, endometrial hyperplasia/dysplasia); (2) some were consistent with the indirect mechanism (ovarian/oviductal salpingitis, cystic ovarian follicles); (3) some were consistent with a combination of the direct and indirect mechanisms (altered endocrine status); and (4) the mechanism(s) for one (lack of corpora lutea) was uncertain. This study also generated some surprising observations regarding vaginal estrous assessments as a means to monitor periodicity of ovarian function in the hamster.

To understand whether bone morphogenetic protein plays any role in the formation of primordial follicles in the hamster, we examined the temporal and spatial expression of bone morphogenetic protein receptor (BMPR) mRNA and protein in embryonic (E) 13 through postnatal day (P) 15 ovarian cells and a possible regulation by FSH during the formation of primordial follicles on P8. BMPRIA and BMPRII mRNA levels were significantly higher than that of BMPR1B throughout ovary development. BMPRIA and BMPRII mRNA levels increased significantly on E14 and declined by P5 through P6. Whereas BMPRII mRNA increased again by P7, BMPRIA mRNA levels increased through P8 concurrent with primordial follicle formation. In contrast, BMPRIB mRNA levels increased greater than 10-fold on P7-9, with a further 3-fold increase by P10. BMPR proteins were low in the somatic cells and oocytes on E13 but increased progressively during postnatal development. BMPR expression in somatic cells increased markedly on P8. Whereas BMPRII expression declined by P10 and remained steady thereafter, BMPRIA protein expression fluctuated until P15 when it became low and steady. Overall, BMPRIB immunoreactivity also declined by P10 and then remained low in the interstitial cells through P15. FSH antiserum treatment on E12 significantly attenuated receptor mRNA and protein levels by P8, but equine chorionic gonadotropin replacement on P1 reversed the inhibition. Furthermore, FSH in vitro up-regulated BMPR levels in P4 ovaries. This unique pattern of BMPR expression in the oocytes and somatic cells during perinatal ovary development suggests that BMP may play a regulatory role in primordial follicle formation. Furthermore, FSH may regulate BMP action by modulating the expression of its receptors.

We have previously reported that the expression of Angiotensin II (Ang II) type 1 receptors (AT1R) was increased in the rostral ventrolateral medulla (RVLM) of rabbits with chronic heart failure (CHF) and in the RVLM of normal rabbits infused with intracerebroventricular (ICV) Ang II. The present study investigated whether oxidant stress plays a role in Ang II–induced AT1R upregulation and its relationship to the transcription factor activator protein 1 (AP1) in CHF rabbits and in the CATHa neuronal cell line. In CATHa cells, Ang II significantly increased AT1R mRNA by 123±11%, P P P P P P P P 3 versus 41.95×10 3 gray level P

Primordial follicles (PF) are formed when somatic cells differentiate into flattened pregranulosa cells, invaginate into the oocyte nests and encircle individual oocytes. We hypothesize that BMP2 regulates PF formation by promoting the transition of germ cells into oocytes and somatic cells into pregranulosa cells. E15 hamster ovaries were cultured for 8 days corresponding to postnatal day 8 (P8) in vivo, with or without BMP2 and the formation of PF was examined. BMP2 was expressed in the oocytes as well as ovarian somatic cells during development. BMP2 exposure for the first two days or the last two days or the entire 8 days of culture led to increase in PF formation suggesting that BMP2 affected both germ cell transition and somatic cell differentiation. Whereas an ALK2/3 inhibitor completely blocked BMP2-induced PF formation, an ALK2-specific inhibitor was partially effective, suggesting that BMP2 affected PF formation via both ALK2 and ALK3. BMP2 also reduced apoptosis in vitro. Further, more meiotic oocytes were present in BMP2 exposed ovaries. In summary, the results provide the first evidence that BMP2 regulates primordial follicle formation by promoting germ cell to oocyte transition and somatic cell to pre-granulosa cells formation and it acts via both ALK2 and ALK3.

The objective was to reveal whether a protein kinase C (PKC [all isozymes])-mediated self-sustaining MAPK3/1 (3/1 extracellular signal regulated kinase 2/1, also known as ERK2/1) activation loop was necessary for FSH- or epidermal growth factor (EGF)-induced DNA synthesis in the granulosa cells of intact preantral follicles. For this purpose, hamster preantral follicles were cultured with FSH or EGF in the presence of selective kinase inhibitors FSH or EGF phosphorylated RAF1, MAP2K1, and MAPK3/1. However, a relatively higher dose of EGF was necessary to sustain the MAPK3/1 activity, which was essential for cyclin-dependent kinase 4 (CDK4) activation and DNA synthesis. In intact preantral follicles, FSH or EGF stimulated DNA synthesis only in the granulosa cells. Sustained activation of MAPK3/1 beyond 3 h was independent of EGFR kinase activity but dependent on PKC activity, which appeared to form a self-sustaining MAPK3/1 activation loop by activating RAF1, MAP2K1, and PLA2G4 (phospholipase A2 [all cytosolic isozymes]). Inhibition of PKC activity as late as 4 h after the administration of FSH or EGF arrested DNA synthesis, which corresponded with attenuated phosphorylation of RAF1 and MAPK3/1, thus suggesting an essential role of PKC in MAPK3/1 activation. Collectively, these data present a novel self-sustaining mechanism comprised of MAPK3/1, PLA2G4, PKC, and RAF1 for CDK4 activation leading to DNA synthesis in granulosa cells. Either FSH or EGF can activate the loop to activate CDK4 and initiate DNA synthesis; however, consistent with our previous findings, FSH effect seems to be mediated by EGF, which initiates the event by stimulating EGFR kinase.

EGF or TGFB1 alone stimulates but together attenuate granulosa cell DNA synthesis. Intact preantral follicles from hamsters were cultured with TGFB1, EGF, or both to reveal the mechanisms of such unique regulation. Follicular CCND2 (also known as cyclin D2), CDKN1B (also known as p27(kip1)), and the involvement of appropriate signaling intermediaries and kinases were examined. TGFB1, acting via SMAD2 and SMAD3, antagonized the degradation of CCND2 protein by blocking its phosphorylation. In contrast, TGFB1 supported CDKN1B degradation by involving MAPK14 (also known as p38 Map Kinase) and PKC, resulting in CDK4 activation and DNA synthesis. EGF via MAPK3/1 maintained functional levels of CCND2 through CCND2 synthesis as well as degradation. EGF and TGFB1 together inhibited CDK4 activation and DNA synthesis. EGF attenuated TGFB1 stimulated phosphorylation of SMAD3, TGFB1-induced activation of MAPK14 and PKC, and TGFB1 suppression of CCND2 degradation. In contrast, TGFB1 suppressed EGF-induced increase in CCND2 mRNA levels. The final outcome was CCND2 degradation without replenishment and decreased activities of MAPK14 and PKC leading to suppression of CDK4 activation. The results indicate that each growth factor involves a separate mechanism to maintain an effective level of CCND2 in granulosa cells for the activation of CDK4 and induction of DNA synthesis. However, their simultaneous action is inhibitory to follicular DNA synthesis because they counteract each other's activity by interfering at specific sites. Because both EGF and TGFB1 are present in granulosa cells, this mechanism may explain how their effects are temporally modulated for granulosa cell proliferation and folliculogenesis.

Postnatal growth differentiation factor 9 (GDF-9) expression in the hamster oocytes precedes the formation of primordial follicles. We examined the functional significance of GDF-9 in primordial folliculogenesis in the hamster ovary using RNA interference knockdown of GDF-9 mRNA and protein expression. Fifteen-day-old fetal ovaries were cultured for 9 d with or without 1 ng FSH, 1 μl Metafectane, 100 nm control nontargeting small interfering RNA (siRNA), GDF-9 siRNA, or GDF-9 siRNA + FSH, and the development of primordial follicles examined. The efficiency of siRNA transfecting ovarian cells in the organ culture was tested by culturing ovaries with siGlo, a nontargeting control siRNA labeled with Cy3. More than 90% of cells in the ovary were siGlo positive, and neither the Metafectane nor the siRNA-induced cellular apoptosis. Control siRNA did not affect the basal levels of GDF-9 mRNA, but GDF-9 siRNA slightly but significantly reduced the level. FSH markedly up-regulated the levels of GDF-9 mRNA and protein, and the effect was completely suppressed by GDF-9 siRNA. However, GDF-9 siRNA did not affect the levels of bone morphogenetic protein receptor IA or β-actin mRNA. GDF-9 siRNA alone also reduced GDF-9 protein expression. Concurrent with GDF-9 expression, FSH significantly augmented primordial follicle formation, but the effect was abolished by GDF-9 siRNA. These results suggest that endogenous GDF-9 plays an important role in somatic cell differentiation and the formation of primordial follicles. Furthermore, FSH, by virtue of regulating GDF-9 expression, modulates oocyte regulation of primordial follicles formation.

Exposure to testosterone (T) during d 30–90 of fetal life results in low-birth-weight offspring, hypergonadotropism, multifollicular ovaries, and early cessation of cyclicity. The multifollicular phenotype may result from failure of follicles to regress and consequent follicular persistence or, alternatively, increased follicular recruitment. We tested the hypothesis that prenatal exposure to excess T causes intrauterine growth retardation and increases ovarian follicular recruitment. Time-mated pregnant ewes were treated with 100 mg T propionate in cottonseed oil or vehicle twice weekly from d 30–90 of gestation. Ewes were euthanized near term, from d 139–141 of gestation (term is 147 d). After determining fetal measures and organ weights, ovaries were removed from fetuses of control and T-treated dams, and follicular distribution in each ovary was determined by morphometric quantification. Total number and percentage distribution of the various classes of follicles (primordial, primary, preantral, and antral follicles) were compared between treatment groups. Prenatally T-treated female fetuses were smaller in size, had an increased head circumference to fetal weight ratio (P < 0.01), increased adrenal to fetal weight ratio (P < 0.05), decreased number of follicles (P < 0.05), a decrease in percentage of primordial follicles (P < 0.001), and a corresponding increase in the remaining classes of follicles (P < 0.05). Ovarian findings support decreased ovarian reserve and enhanced follicular recruitment, potential contributors of early reproductive failure. The extent to which metabolic changes associated with intrauterine growth retardation contribute toward altered trajectory of ovarian folliculogenesis remains to be determined.

Although gonadotropins have been reported to downregulate FSH-receptor (FSHR) mRNA levels in the ovaries of female rats, the effect of the gonadotropin surge, particularly FSH, on hamster follicular FSHR mRNA levels warrants further examination. The objectives of the present study were to clone and determine the complete FSHR cDNA sequence of the hamster and to delineate the effects of endogenous and exogenous FSH on the steady-state levels of ovarian FSHR mRNA. Complete FSHR cDNA was derived from hamster ovarian total RNA by the strategy of 3'- and 5'-rapid amplification of cDNA ends. Ovaries were obtained before and after the endogenous gonadotropin surge or exogenous FSH administration, and the steady-state levels of FSHR mRNA were assessed by Northern blot hybridization. Cloned FSHR cDNA consists of a reading frame corresponding to exons 1-10 of the human FSHR gene and the 5'- and 3'-untranslated regions. The nucleic acid and amino acid sequences of the reading frame were at least 87% and 92% identical, respectively, to that of human, rat, and mouse FSHR. Furthermore, the amino acid sequence contained seven transmembrane domains characteristic of the FSHR. The steady-state levels of FSHR mRNA increased from estrus (Day 1) to reach a peak on proestrus (Day 4) noon; however, significant attenuation was noted following the gonadotropin surge, which was blocked by phenobarbital. Exogenous FSH also downregulated, both dose- and time-dependently, ovarian FSHR mRNA levels. These data indicate that the nucleic acid sequence of hamster FSHR has been identified and that FSH modulates FSHR mRNA levels in the hamster ovary.

Growth differentiation factor-9 (GDF-9) and stem cell factor (SCF) influence follicle formation beyond the primary stage; however, factors influencing the formation of primordial follicles remain elusive. To determine whether GDF-9 and SCF promoted primordial follicle formation during ovarian morphogenesis in the hamster, and whether FSH had any modulatory influence, fetal ovaries were collected on Gestation Day 15 from pregnant hamsters treated with or without an FSH antiserum on Gestation Day 12 and cultured in vitro up to Day 9 with SCF, GDF-9, or FSH. The percentages and diameters of primordial, primary, and secondary follicles and their oocytes were determined by morphometric evaluation, and the expression of GDF-9 was detected by immunolocalization. SCF, GDF-9, and FSH promoted primordial and primary follicle formation, but GDF-9 was more efficient. The diameters of the follicles developed under GDF-9 or FSH, but not SCF, compared well with those developed in vivo. FSH- and GDF-9-induced folliculogenesis was attenuated by the SCF antibody. Similarly, in vitro formation of primordial follicles decreased markedly in ovaries exposed to the FSH antiserum in utero, which was reversed by SCF, GDF-9, or FSH; however, GDF-9 had a profound effect on follicular development. GDF-9 protein appeared exclusively in the oocytes on Postnatal Day 4; however, it appeared in vitro by 48 h, and the expression was upregulated by FSH. These results suggest that although SCF-induced primordial follicle formation constitutes primarily somatic cell development, GDF-9 influences both the oocyte and its companion somatic cells. FSH plays an important role in primordial folliculogenesis in the hamster via GDF-9 and SCF.

Although cyclin D2 mRNA synthesis precedes gonadotropin-induced DNA synthesis in quiescent granulosa cells in culture, it is unclear whether a similar mechanism exists for the granulosa cells of growing preantral follicles in cyclic animals. The objective was to evaluate whether the synthesis of cyclin D2 protein was a prerequisite for FSH-induced DNA synthesis in the granulosa cells of intact preantral follicles of cyclic hamsters. Preantral follicles from cyclic hamsters were cultured in the presence or absence of FSH, and cell cycle parameters were examined. FSH stimulated cyclin-dependent kinase (CDK)-4 activity by 2 h and DNA synthesis by 4 h without altering the levels of cyclin D2 in the granulosa cells. The FSH effect was mimicked by epidermal growth factor administered in vivo. Although FSH increased the levels of cyclin D2 mRNA, it also stimulated the degradation of cyclin D2 as well as p27(Kip1) and p19(INK4) proteins. FSH activation of CDK4 was mediated by cAMP and ERK-1/2. In contrast to granulosa cells in intact follicles, FSH or cAMP significantly increased cyclin D2 protein levels in cultured granulosa cells but failed to induce DNA synthesis. Collectively, these data suggest that granulosa cells of preantral follicles, which are destined to enter the S phase during the estrous cycle, contain necessary amounts of cyclin D2 and other G1 phase components. FSH stimulation results in the formation and activation of the cyclin D2/CDK4 complex leading to DNA synthesis. This mechanism may be necessary for rapid movement of follicles from preantral to antral stages during the short duration of the murine estrous cycle.

To evaluate the site(s) and mechanism(s) of glucocorticoid-inhibition of transforming growth factor (TGF) beta receptor (TbetaR) mRNA expression in ovarian cells, steady-state levels of TbetaR mRNA in hamster preantral follicles exposed to FSH or estradiol with or without dexamethasone were determined by reverse transcription polymerase chain reaction and Southern hybridization. The effect of dexamethasone on follicular DNA and steroid synthesis and the expression of NF-Y and Sp3 were also investigated. Dexamethasone differentially inhibited FSH- or estradiol-induced expression of TbetaR mRNA in preantral follicles at all stages. Dexamethasone also strongly inhibited FSH-induced but not TGFbeta2-induced follicular DNA synthesis, and the inhibition was completely reversed by TGFbeta2. However, TGFbeta2 markedly attenuated FSH + dexamethasone-stimulated progesterone and FSH-induced follicular estradiol synthesis. Both FSH and estradiol upregulated NF-YA expression, but the effect was significantly attenuated by dexamethasone. Our results suggest that suppression of NF-YA levels is one of the mechanisms whereby dexamethasone reduces hormone-induced TbetaRI and TbetaRII mRNA levels in hamster preantral follicles. Dexamethasone potentiates the effect of FSH on granulosa cell steroidogenesis, whereas TGFbeta counteracts the effect. These data indicate that glucocorticoid and TGFbeta may form an important regulatory loop to modulate FSH regulation of preantral follicular growth and differentiation.

Spatiotemporal expression, endocrine regulation, and activation of epidermal growth factor receptor (EGFR) in the hamster ovary were evaluated by immunofluorescence and in situ hybridization localization. Whereas granulosa cells (GC) of primordial through large preantral (stage 6, 7-8 layers GC) follicles had low immunoreactivity, granulosa cells of antral follicles, theca, and interstitial cells had intense EGFR immunoreactivity. EGFR expression in GC of primordial and small preantral follicles increased progressively from estrous through proestrous, but a significant increase occurred in mural GC of antral follicles following the gonadotropin surge. Interstitial cells around small preantral follicles had strong immunofluorescence, and the intensity increased significantly in fully differentiated thecal cells. Distinct EGFR protein was localized in the nucleus of the oocytes and granulosa cells. FSH significantly stimulated EGFR expression in the GC, especially the mural GC, theca, and interstitial cells in hypophysectomized hamster. Estrogen stimulated EGFR expression in preantral GC as well as in interstitial cells. Progesterone and hCG effect was limited to theca and interstitial cells. EGFR expression correlated well with EGFR activation following endogenous or exogenous gonadotropin exposure. Receptor mRNA expression closely followed the protein expression, with increased mRNA expression in mural GC of antral follicles. These results suggest that low levels of EGF signal as a consequence of low levels of receptors in preantral GC may be critical for cell proliferation, but higher receptor density may evoke increased signal intensity due to activation of other intracellular signal pathways, which activate cellular processes related to granulosa, theca, and interstitial cell differentiation. The spatiotemporal cell type and follicle stage-specific expression of receptor mRNA and protein and EGFR activation is critically regulated by gonadotropins and ovarian steroids, primarily estradiol.

Heparin binding EGF-like growth factor (HB-EGF), encoded by the Hegfl gene, is considered as an important mediator of embryo-uterine interactions during implantation in mice. However, it is unknown whether HB-EGF is important for implantation in species with different steroid hormonal requirements. In mice and rats, maternal ovarian estrogen and progesterone (P4) are essential to implantation. In contrast, blastocyst implantation can occur in hamsters in the presence of P4 alone. To ascertain whether HB-EGF plays any role in implantation in hamsters, we examined the expression, regulation and signaling of HB-EGF in the hamster embryo and uterus during the periimplantation period. We demonstrate that both the blastocyst and uterus express HB-EGF during implantation. Hegfl is expressed solely in the uterine luminal epithelium surrounding the blastocyst prior to and during the initiation of implantation. Hypophysectomized P4-treated pregnant hamsters also showed a similar pattern of implantation-specific Hegfl expression. These results suggest that uterine Hegfl expression at the implantation site is driven by either signals emanating from the blastocyst or maternal P4, but not by maternal estrogen. However, in ovariectomized hamsters, uterine induction of Hegfl requires the presence of estrogen and activation of its nuclear receptor (ER), but not P4. This observation suggests an intriguing possibility that an estrogenic or unidentified signal from the blastocyst is the trigger for uterine HB-EGF expression. An auto-induction of Hegfl in the uterus by blastocyst-derived HB-EGF is also a possibility. We further observed that HB-EGF induces autophosphorylation of ErbB1 and ErbB4 in the uterus and blastocyst. Taken together, we propose that HB-EGF production and signaling by the blastocyst and uterus orchestrate the ‘two-way’ molecular signaling to initiate the process of implantation in hamsters.

Spatiotemporal expression patterns of ER-alpha and ER-beta protein and mRNA in hamster ovarian cells during the estrous cycle and following hypophysectomy and selective hormone replacement were evaluated by immunofluorescence, immunoblotting and in situ hybridization analyses. Whereas ER-beta mRNA and protein expression predominated in granulosa cells and ER-alpha expression was in interstitial and thecal cells, overlap in receptor subtype expression across cell types was evident. Both ER subtypes were present from primordial follicle stage onward. ER-alpha mRNA levels and immunoreactivity started increasing from D3:0900 h in interstitial and granulosa cells and peaked on the proestrous (D4:0900 h). Regionalized higher expression of ER-alpha in granulosa cells in and around the forming antrum was evident. Surface epithelial cells were also positive. ER-beta mRNA and protein expression increased markedly in granulosa and interstitial cells on D2:0900 h, reached a peak on D3:0900 h, and then declined sharply on D4:0900 h. No change in ER expression occurred following the preovulatory gonadotropin surge. Whereas FSH or human CG stimulated ER-alpha mRNA and protein expression in hypophysectomized hamsters, only FSH could stimulate ER-beta mRNA and protein, and the effect was significantly attenuated by human CG. ER expression was stimulated by estrogen, but progesterone strongly inhibited estrogen action. These results indicate that ER expression is cell type specific to the larger extent and is critically regulated by reproductive hormones.

FSH plays an important role in ovarian follicular development, and it functions via the G-protein coupled FSH receptor. The objectives of the present study were to determine if full-length FSHR mRNA and corresponding protein were expressed in fetal through postnatal hamster ovaries to explain the FSH-induced primordial follicle formation, and if FSH or estrogen (E) would affect the expression. A full-length and two alternately spliced FSHR transcripts were expressed from E14 through P20. The level of the full-length FSHR mRNA increased markedly through P7 before stabilizing at a lower level with the formation and activation of primordial follicles. A predicted 87 kDa FSHR protein band was detected in fetal through P4 ovaries, but additional bands appeared as ovary developed. FSHR immunosignal was present in undifferentiated somatic cells and oocytes in early postnatal ovaries, but was granulosa cells specific after follicles formed. Both eCG and E significantly up-regulated full-length FSHR mRNA levels. Therefore, FSHR is expressed in the hamster ovary from the fetal life to account for FSH-induced primordial follicle formation and cAMP production. Further, FSH or E regulates the receptor expression.

Epidermal growth factor (EGF) is mitogenic to preantral follicles, and transforming growth factor β (TGFβ) influences ovarian cell functions in a variety of species. Although an interaction of these ligands during preantral folliculogenesis is likely, whether EGF influences TGFβ action on preantral follicles by modulating TGFβ receptor (TβR) gene transcription and translation is not known. To determine whether EGF influenced TβR mRNA and protein levels in granulosa cells during preantral folliculogenesis, hamster preantral follicles at stages 1–6 were cultured in the absence or presence of EGF and follicular TβR mRNA, and protein levels were monitored by semiquantitative reverse transcription polymerase chain reaction and immunoblotting, respectively. Both TβR type I (TβRI) and TβR type II (TβRII) mRNA and protein were present in preantral follicles, and their expression was up-regulated by EGF in a stage-dependent manner. However, EGF effect on the expression of TβRI and TβRII was differential. ...

Whereas FSH action is critical for the growth of preantral follicles, its role in the development of primordial follicles is controversial. The objective of the present study was to evaluate whether perinatal (fetal through early postnatal) FSH action is needed for the formation of primordial follicles, which first appear in the hamster ovary on the 7th to 8th day of postnatal life. A single dose of FSH-specific polyclonal antibody was injected into pregnant hamsters on the 12th, 13th, or 14th day of gestation and into newborn hamsters. Some of the antibody-exposed postnatal hamsters were injected with a single dose of equine CG (eCG) to check the reversibility of the antibody action. Ovaries were collected on D8pn or D12pn, and the percentage of primordial, primary, and secondary follicles was quantitated morphometrically. Ovaries of 8-day-old hamsters that were born to mothers treated with a single sc dose of the anti-FSH-antibody on day 12 of gestation had significantly reduced numbers of primordial fo...

The temporal and spatial expression of cytochrome P450 side-chain cleavage (CYP11A1) and cytochrome P450 17 alpha-hydroxylase (CYP17) mRNA and protein during thecal cell differentiation in developing hamster ovaries were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence histochemistry, respectively. Ovaries were collected from 15-day fetal through 20-day-old postnatal hamsters and used either for immunofluorescence detection of enzyme protein or RT-PCR evaluation of enzyme mRNA. Immunoreactivity of CYP11A1 first appeared in the interstitial cells on Day 10 postnatal (PN), and the intensity increased significantly with further ovarian development beyond 11 days of age. In contrast, CYP17 immunostaining was first detected in a few interstitial cells closer to large preantral follicles by Day 12 PN, and their number increased appreciably by Day 14 PN. By age 18-20 days, CYP17-positive cells were localized primarily in the thecal layer of large preantral follicles. A low level of CYP17 and CYP11A1 mRNA was present in fetal ovaries. The CYP17 mRNA levels increased sharply by Day 1 PN but decreased to a low baseline level by Day 2 PN and remained low up to Day 9 PN. Both CYP11A1 and CYP17 mRNA levels increased significantly by Day 10 PN compared to Day 9 PN; however, the increase for CYP11A1 was greater than CYP17. The CYP11A1 mRNA levels decreased noticeably on Day 11 PN and remained relatively stable until Day 14 PN; however, mRNA levels started increasing by Day 15 PN and increased sharply by Day 17 PN onward, corresponding to the increase in CYP11A1 protein in the ovarian interstitium and thecal compartments. On the other hand, CYP17 mRNA expression increased progressively through Day 12 PN. A sharp increase in CYP17 mRNA was noted on Day 13 PN in conjunction with the morphological development of thecal cells; mRNA levels remained steady afterward. The correlation of the increase in enzyme mRNA and protein, especially of CYP17, with the morphological development of thecal layers suggests that the differentiation of interstitial cells into theca may be modulated by multilayered preantral follicles, and the expression of enzyme protein occurs prior to an increase in serum LH.

Interstitial cells in the neonatal hamster do not respond to LH in vitro; however, side-chain cleavage (CYP11A1) and 17alpha-hydroxylase (CYP17) enzyme proteins are expressed in these cells. The objective of the study was to evaluate whether the cAMP second messenger system was active in these cells and if cAMP upregulates the levels of CYP11A1 and CYP17 mRNA. Interstitial cells (ICs) were cultured for 96 h in the presence of 5% fetal bovine serum and then cultured in serum-free medium in the presence of LH, forskolin, or 8-Br-cAMP for 24 h. The accumulation of cAMP, progesterone, and androstenedione was measured by radioimmunoassay, whereas CYP11A1 and CYP17 mRNA levels were determined by a semiquantitative reverse transcription-polymerase chain reaction and Southern hybridization analysis. LH failed to induce either progesterone or androstenedione production; however, forskolin stimulated cAMP production by interstitial cells in a dose-dependent manner. Moreover, both forskolin and 8-Br-cAMP significantly elevated the levels of CYP11A1 and CYP17 mRNA and induced progesterone synthesis by the interstitial cell monolayer. Despite the increase in CYP17 mRNA levels by 8-Br-cAMP, no appreciable change was noted in androstenedione production. These results suggest that, in vitro, a fully functional adenylate cyclase system is present in cultured interstitial cells of the neonatal hamster and that cAMP can influence the expression of CYP11A1 and CYP17 genes; however, cultured cells do not appear to express LH receptors that are functionally linked to the adenylate cyclase system. Moreover, the translation of CYP17 mRNA may require additional factors, which may originate from maturing granulosa cells.

The hormonal regulation of ovarian transforming growth factor-beta (TGF-beta) type I receptor (TbetaRI) and TbetaRII messenger (mRNA) expression was evaluated using cyclic and hypophysectomized hamsters. Northern blot analysis revealed that three TbetaRI and one TbetaRII gene transcripts were expressed in the hamster ovary. Reverse transcription-polymerase chain reaction quantitation revealed that receptor mRNA was differentially expressed during the estrous cycle. Although, mRNA levels for both receptor types increased steadily up to Day 4:0900 h, a sharp decline occurred following the gonadotropin surge. In fact, receptor mRNA started declining by Day 4:1200 h, long before the gonadotropin surge; however, only TbetaRI mRNA levels recovered partially by 1500 h to fall again by 1600 h. Although hypophysectomy preferentially reduced TbetaRII mRNA levels, gonadotropins as well as ovarian steroids significantly induced TbetaRI and TbetaRII mRNA expression within 48 h and 24 h, respectively; 5alpha-dihydrotesterone (DHT) induced only TbetaRII mRNA. The induction of ovarian TbetaRI and TbetaRII mRNA by estradiol-17beta() or progesterone was severely attenuated by dexamethasone. A marked increase in serum cortisol coincided with the periovulatory rise in serum gonadotropins. These results suggest that the increase in TGF-beta receptor mRNA expression correlates with gonadotropin-induced ovarian follicular development during the estrous cycle. Moreover, receptor mRNA expression is critically and differentially regulated by gonadotropins as well as ovarian steroids. Most importantly, glucocorticoid appears to play a critical modulatory role in the temporal expression of receptor mRNA in the ovary, hence, controlling folliculogenesis.

Fetal hamster ovaries were cultured for up to 16 days in the presence or absence of various dosages of insulin to evaluate the induction of folliculogenesis in vitro. In the absence of insulin, a few primordial follicle-like structures appeared by the 4th day, and distinct primary follicles (stage 1) appeared by the 12th day of culture. The organelles in the oocytes and adjacent granulosa cells developed along with follicular growth. Moreover, gap junctions between the oocyte and somatic cell plasma membrane also developed as early as 8 days in culture. In the presence of 0.2 microg/ml insulin, primary follicles developed after 8 days, and approximately 4% secondary follicles with 2-3 layers of granulosa cells appeared after 16 days of culture. However, higher dosages (> 0.2 microg/ml) of insulin retarded primary follicle formation and induced the formation of primordial follicles with larger oocytes. An increased number of larger oocytes with a few granulosa cells accumulated at the periphery of the ovary. The results indicate that although primordial and primary follicles can develop after 12 days in vitro in the absence of exogenous insulin, the latter is required for timely progression of follicular development through primary and secondary stages.

Modulation of glucose metabolic capacity of human preantral follicles in vitro by gonadotropins and intraovarian growth factors was evaluated by monitoring the activities of phosphofructokinase (PFK) and pyruvate kinase (PK), two regulatory enzymes of the glycolytic pathway, and malate dehydrogenase (MDH), a key mitochondrial enzyme of the Krebs cycle. Preantral follicles in classes 1 and 2 from premenopausal women were cultured separately in vitro in the absence or presence of FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF-I), or transforming growth factor beta1 (TGFbeta1) for 24 h. Mitochondrial fraction was separated from the cytosolic fraction, and both fractions were used for enzyme assays. FSH and LH significantly stimulated PFK and PK activities in class 1 and 2 follicles; however, a 170-fold increase in MDH activity was noted for class 2 follicles that were exposed to FSH. Although both EGF and TGFbeta1 stimulated glycolytic and Krebs cycle enzymes for class 1 preantral follicles, TGFbeta1 consistently stimulated the activities of both glycolytic enzymes more than that of EGF. IGF-I induced PK and MDH activities in class 1 follicles but negatively influenced PFK activity for class 1 follicles. In general, only gonadotropins consistently stimulated both glycolytic and Krebs cycle enzyme activities several-fold in class 2 follicles. These results suggest that gonadotropins and ovarian growth factors differentially influence follicular energy-producing capacity from glucose. Moreover, gonadotropins may either directly influence glucose metabolism in class 2 preantral follicles or do so indirectly through factors other than the well-known intraovarian growth factors. Because growth factors modulate granulosa cell mitosis and functionality, their role on energy production may be related to specific cellular activities.

A possible relationship between transforming growth factor beta receptor type I (TbetaRI) and type II (TbetaRII) protein expression in human granulosa cells and the quality of preimplantation embryo development in vitro was studied using immunoblot analysis of TbetaRI and TbetaRII in hyperstimulated granulosa cells and morphological assessment of the cleavage potential of the zygotes in vitro. Washed granulosa cells were collected from

The function of ovarian interstitial cells has been largely addressed using rat theca-interstitial cell culture. However, this preparation is primarily enriched with theca and secondary interstitial cells, which make it difficult to address selectively the function of the primary interstitial cells. We have developed an in vitro culture of hamster ovarian primary interstitial cells. Cells were isolated from postnatal hamster ovaries by collagenase digestion and purified over a Percoll gradient. The preparation contained 90% viable, pure interstitial cells, which anchored to the plastic and glass culture surface in the presence of fetal bovine serum. Cell proliferation was noted in the presence of serum dosages higher than 0.2%; however, reduction of serum concentration to 0.1% or complete serum starvation did not affect cell viability but almost completely abolished cell proliferation as determined by [3H]thymidine incorporation, labeling index, and DNA content of the culture. All cells exhibited active 3beta-hydroxysteroid dehydrogenase and P450 side chain cleavage immunoreactivity, which corresponded to basal progesterone and androstenedione accumulation. Replacement of serum to starving cells resulted in the induction of the "S" phase and "M" phase specific cyclins, and resumption of cell proliferation. Our results indicate that hamster primary interstitial cells can be cultured in vitro as a monolayer, and the anchorage and proliferation of these cells depend on serum supplement; however, a viable monolayer can be maintained for several days without serum. This model will be useful for addressing the mechanisms of differentiation of ovarian interstitial cells.

Estradiol-17β (E) plays an important role in ovarian follicular development. Evidence indicates that some of the effect of E is mediated by the transmembrane estrogen receptor. In this study, we examined the spatio-temporal expression of recently discovered ERα36 (ESR36), a splice variant of Esr1 and a receptor for non-genomic E signaling, in the hamster ovary during the estrous cycle and the role of gonadotropins and ovarian steroid hormones in ESR36 expression. ESR36 expression was high on estrus (D1:0900 h) and declined precipitously by proestrus (D4:0900 h) and remained low up to D4:1600 h. Immunofluorescence findings corroborated immunoblot findings and revealed that ESR36 was expressed only in the cell membrane of both follicular and non-follicular cells, except the oocytes. Ovarian ESR36 was capable of binding to the E-affinity matrix, and have different molecular weight than that of the ESR1 or GPER. Hypophysectomy (Hx) resulted in a marked decline in ESR36 protein levels. FSH and LH, alone or combined, markedly upregulated ESR36 protein in Hx hamsters to the levels observed in D1 hamsters, but neither E nor P had any effect. Inhibition of the gonadotropin surge by phenobarbital treatment on D4:1100 h attenuated ESR36 expression in D1:0900 h ovaries, but the decline was restored by either FSH or LH replacement on D4 afternoon. This is the first report to show that ESR36, which is distinct from ESR1 or GPER is expressed in the plasma membrane of ovarian follicular and non-follicular cells, binds to E and its expression is regulated directly by the gonadotropins. In light of our previous findings, the results suggest that ovarian cells contain at least two distinct membrane estrogen receptors, such as GPER and ESR36, and strongly suggest for a non-genomic action of E regulating ovarian follicular functions.

The hormonal regulation of transforming growth factor-beta (TGF beta) receptor type II (T beta RII) protein expression in the hamster ovary was evaluated by [125I]TGF beta 1 cross-linking, immunolocalization, and immunoprecipitation of T beta RII using receptor-specific antibodies. Granulosa cells of preantral and antral follicles, and interstitial and luteal cells showed strong signal on day 1 at 0900 h; interstitial and luteal staining was maximum. Immunoreactivity in the interstitium fell by day 2 and reappeared on day 4. A sharp reduction of immunostaining occurred after the gonadotropin surge. In hypophysectomized hamsters, FSH induced T beta RII in both granulosa and interstitial cells, but LH was effective only on interstitial cells. Whereas 17 beta-estradiol (17 beta E2) efficiently induced interstitial T beta RII, progesterone severely attenuated E2 induction of receptor protein. Apart from a membrane-associated form of T beta RII, a novel cytosolic form of T beta RII was detected. The cytosolic T beta RII is a glycoprotein with N'-linked oligosaccharides, like its membrane counterpart, possessing serine-threonine kinase activity that is TGF beta sensitive. The membrane-associated T beta RII disappeared on day 2 of the cycle, but reappeared by day 4 in the morning. A good correlation was found with the cytosolic form. Hypophysectomy diminished, whereas FSH, LH, and 17 beta E2 increased both forms of T beta RII; however, LH induction of the cytosolic form was greater than that by FSH. Progesterone prevented T beta RII protein integration to the membrane, but testosterone and dihydrotestosterone were also effective in T beta RII induction in the membrane. A high E2/progesterone ratio was an important determinant in T beta RII induction in the cell membrane. These results provide the first direct evidence for the presence of a functional T beta RII in the ovary. The receptor is a serine/threonine kinase and exists as distinct cytosolic and membrane forms that show a unique relationship during the estrous cycle. The induction of ovarian T beta RII is critically and temporally influenced by gonadotropins and ovarian steroid hormones.

Follicular development in the mammalian ovary is a complex process regulated by an orchestrated action of the pituitary gonadotropins, e.g., follicle stimulating hormone (FSH) and luteinizing hormone (LH), and local ovarian factors, such as peptide growth factors and steroids. The mechanism of endocrine/paracrine/autocrine regulation of folliculogenesis (i.e., cell proliferation and functions) has been addressed largely by biochemical means. However, the availability of immunological and molecular tools now enables us to undertake critical microscopic studies revealing the ovarian cell-type specific synthesis and/or accumulation of many of these local peptide modulators, their roles in the proliferation and differentiation of follicular cells, and their regulation by gonadotropins and local factors. The objective of this review is to provide a comprehensive yet complete picture of the endocrine/autocrine regulation of mammalian folliculogenesis as revealed by microscopic studies. Efforts have been made to include adequate research information relevant to update our understanding of the process of follicular development; however, to maintain the brevity, many equally important studies could not be included. This review confirms that FSH and LH are still the primary stimuli for follicular development. However, it is clear that the actions of these hormones at the cell level involve a host of peptide factors which are produced locally by different follicular cell types and which are powerful modulators of gonadotropin actions. The temporal and spatial expression of the genes of these modulators, the synthesis of active factors, their interactions, and the dynamics of their receptors on the follicular cell surface may be the ultimate determinants of cellular events which are crucial to coordinated growth and differentiation of follicular cells leading to folliculogenesis and ovulation.

Follicular development commences with the formation of primordial follicles, which begins with the differentiation of pluripotent ovarian somatic cells into early granulosa cells and their apposition to the oocytes in the egg nest. The process of primordial follicle morphogenesis and factors affecting the formation and development of primordial follicles can be examined in vitro using fetal ovaries in organ culture. The functions of candidate genes involved in primordial folliculogenesis can be examined using siRNA or shRNA, which can knockdown specific mRNA targets at specific time points. Here, we describe the organ culture protocol for fetal hamster ovary with GPR30 siRNA as an example. The method to morphologically analyze follicular development is also discussed.

Follicular development commences with the formation of primordial follicles, which begins with the differentiation of pluripotent ovarian somatic cells into early granulosa cells and their apposition to the oocytes in the egg nest. The process of primordial follicle morphogenesis and factors affecting the formation and development of primordial follicles can be examined in vitro using fetal ovaries in organ culture. The functions of candidate genes involved in primordial folliculogenesis can be examined using siRNA or shRNA, which can knockdown specific mRNA targets at specific time points. Here, we describe the organ culture protocol for fetal hamster ovary with GPR30 siRNA as an example. The method to morphologically analyze follicular development is also discussed.

Although many proteins have been shown to affect the transition of primordial follicles to the primary stage, factors regulating the formation of primordial follicles remains sketchy at best. Differentiation of somatic cells into early granulosa cells during ovarian morphogenesis is the hallmark of primordial follicle formation; hence, critical changes are expected in protein expression. We wanted to identify proteins, the expression of which would correlate with the formation of primordial follicles as a first step to determine their biological function in folliculogenesis. Proteins were extracted from embryonic (E15) and 8-day-old (P8) hamster ovaries and fractionated by two-dimensional gel electrophoresis. Gels were stained with Proteosilver, and images of protein profiles corresponding to E15 and P8 ovaries were overlayed to identify protein spots showing altered expression. Some of the protein spots were extracted from SyproRuby-stained preparative gels, digested with trypsin, and analyzed by mass spectrometry. Both E15 and P8 ovaries had high molecular weight proteins at acidic, basic, and neutral ranges; however, we focused on small molecular weight proteins at 4–7 pH range. Many of those spots might represent post-translational modification. Mass spectrometric analysis revealed the identity of these proteins. The formation of primordial follicles on P8 correlated with many differentially and newly expressed proteins. Whereas Ebp1 expression was downregulated in ovarian somatic cells, Sfrs3 expression was specifically upregulated in newly formed granulosa cells of primordial follicles on P8. The results show for the first time that the morphogenesis of primordial follicles in the hamster coincides with altered and novel expression of proteins involved in cell proliferation, transcriptional regulation, and metabolism. Therefore, formation of primordial follicles is an active process requiring differentiation of somatic cells into early granulosa cells and their interaction with the oocytes.

Objective To isolate intact preantral follicles from the human ovary for in vitro studies. Procedure Premenopausal human ovary was digested by a mixture of collagenase and deoxyribonuclease (DNase) for 1 hour at 37°C and for 36 hour at 4°C. Intact preantral follicles at classes 1 and 2 were isolated and cultured for up to 120 hours in a serum-free Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) with ITS + (insulin, transferrin, selenium, linoleic acid, and bovine serum albumin; Collaborative Research, Bedford, MA) with or without FSH. Follicular DNA synthesis was measured by [ 3 H]thymidine incorporation, whereas steroidogenic capacity was assessed by P, androstenedione (A), and 17 β -E 2 RIA. The morphology of long-term cultured follicles was studied by routine histologic procedure. Results A large number of intact, preantral follicles was efficiently dissociated from a portion of ovary. Morphologically, follicles were healthy and did not have any thecal layer. Follicle-stimulating hormone, in vitro, induced follicular DNA synthesis by 24 hours and antrum formation in class 2 follicles after 120 hours. Both class 1 and 2 follicles were able to produce a small basal amount of P and A, but they did not produce any measurable E 2 . However, both classes synthesized increasing amounts of P, A, and E 2 after FSH exposure. Conclusion This is a first report of a successful enzymatic isolation of human preantral follicles and their long-term culture in vitro. The growth and differentiation of preantral follicles by FSH clearly indicate the importance of FSH in human follicular development. These results provide an opportunity for quantitative studies on factors regulating folliculogenesis in the human and a novel future direction for human IVF.

The interactions of epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) in modulating FSH-induced follicular DNA synthesis were studied in isolated intact preantral (stages 1-6) and early antral (stage 7) ovarian follicles from adult hamsters. Follicles were exposed in vitro for 24 h to FSH (100 ng), EGF (50 ng), TGF-beta 1 (1-10 ng), or TGF-beta 2 (1-10 ng), either alone or in combination. The rate of DNA synthesis was assessed by measuring the incorporation of [3H]thymidine into follicular DNA. Both EGF and FSH significantly stimulated follicular DNA synthesis compared with that in controls. Both isoforms of TGF-beta significantly increased follicular [3H]thymidine incorporation in a dose-dependent manner; the effect was greater for small preantral follicles, such as those of stages 1-4. Interestingly, TGF-beta significantly inhibited EGF-induced follicular DNA synthesis, but the rates of DNA synthesis for most of the stages were still higher than that of the control follicles. Specificity of the TGF-beta action on follicular DNA synthesis was evident from the ability of isoform-specific anti-TGF-beta antibodies to neutralize the effect. These antibodies also reversed the TGF-beta inhibition of EGF-induced DNA synthesis. Surprisingly, although TGF-beta attenuated EGF-induced DNA synthesis, it synergized with FSH to stimulate follicular DNA synthesis. Interestingly, FSH-induced DNA synthesis remained unaffected by the anti-TGF-beta antibodies, indicating that TGF-beta may not mediate FSH action on follicular DNA synthesis. These studies suggest that a critical interaction of EGF and TGF-beta modulates granulosa cell proliferation during folliculogenesis in the hamster.(ABSTRACT TRUNCATED AT 250 WORDS)

We examined the expression and hormonal regulation of E-cadherin (CDH1) and N-cadherin (CDH2) with respect to primordial follicle formation. Hamster Cdh1 and Cdh2 cDNA and amino acid sequences were more than 90% similar to those of the mouse, rat, and human. Although CDH1 expression remained exclusively in the oocytes during neonatal ovary development, CDH2 expression shifted from the oocytes to granulosa cells of primordial follicles on postnatal day (P)8. Subsequently, strong CDH2 expression was restricted to granulosa cells of growing follicles. Cdh2 mRNA levels in the ovary decreased from embryonic d 13 through P10 with a transient increase on P7, which was the day before the appearance of primordial follicles. Cdh1 mRNA levels decreased from embryonic d 13 through P3 and then showed a transient increase on P8, coinciding with the formation of primordial follicles. CDH1 and CDH2 expression were consistent with that of mRNA. Neutralization of FSH in utero impaired primordial follicle formation with an associated decrease in Cdh2 mRNA and CDH2, but an increase in Cdh1 mRNA and CDH1 expression. The altered expression was reversed by equine chorionic gonadotropin treatment on P1. Whereas a CDH2 antibody significantly reduced the formation of primordial and primary follicles in vitro, a CDH1 antibody had the opposite effect. This is the first evidence to suggest that primordial follicle formation requires a differential spatiotemporal expression and action of CDH1 and CDH2. Further, FSH regulation of primordial follicle formation may involve the action of CDH1 and CDH2.

Cell-type-specific localization and gonadotropin regulation of transforming growth factor-beta 1 (TGF-beta 1) and transforming growth factor-beta 2 (TGF-beta 2) in the hamster ovary were evaluated immunohistochemically under three conditions: (1) during the estrous cycle (Day 1 = estrus; Day 4 = proestrus); (2) after the blockade of periovulatory gonadotropin surges by phenobarbital, and (3) after FSH and/or LH treatment of long-term hypophysectomized hamsters. Ovarian TGF-beta 1 activity was primarily localized in theca and interstitial cells. The activity increased moderately but significantly after the preovulatory LH surge and reached a peak at 0900 h, Day 2 h; oocytes showed considerable activity. TGF-beta 1 immunoreactivity subsequently fell to low levels in theca-interstitial cells through 0900 h, Day 4. Significant TGF-beta 2 immunoreactivity appeared after the surge, mainly in the granulosa cells of both preantral and antral follicles; a few interstitial cells surrounding preantral follicles showed discrete staining. TGF-beta 2 immunoreactivity in granulosa cells and in interstitial cells next to preantral follicles reached a peak at 0900 h, Day 1, and persisted up to 0900 h, Day 2; oocytes showed no staining. Phenobarbital treatment blocked the appearance of TGF-beta 1 and TGF-beta 2 immunoreactivities at 1600 h, Day 4; however, a rebound in immunoreactivities was observed with the onset of the surge after a 1-day delay. Replacement of LH to long-term hypophysectomized hamsters resulted in a marked increase in TGF-beta 1 immunoreactivity in the interstitial cells, but FSH, although it induced follicular development, did not influence ovarian TGF-beta 1 activity. Treatment with FSH, however, induced a massive increase in TGF-beta 2 immunoreactivity in the granulosa cells of newly developed antral and preantral follicles but not in the interstitial cells; LH, on the other hand, had no significant effect on TGF-beta 2 activity. Treatment with FSH and LH combined resulted in a dramatic increase in TGF-beta 2 immunoreactivity in granulosa and interstitial cells and in TGF-beta 1 in theca and interstitial cells comparable to their peak activity in intact animals. Western analyses substantiated the presence of TGF-beta 1 and TGF-beta 2 in the hamster ovary and the specificity of immunolocalization. These studies, therefore, provide critical evidence that TGF-beta 1 and TGF-beta 2 in the hamster ovary are expressed in specific cell types and that their expression is differentially regulated by LH and FSH, respectively.

The objective of this study was to determine whether surface-modified nanoparticles enhance permeability across nasal mucosa, while retaining the effectiveness of the payload. The uptake and permeability of polystyrene nanoparticles (PS-NPs; FluoSpheres) was evaluated across the various regions of the bovine nasal epithelia following conjugation with deslorelin and transferrin. Uptake and transport of PS-NPs, deslorelin-PS-NPs, and transferrin-PS-NPs exhibited regional differences in the order: inferior turbinate posterior (ITP) > medium turbinate posterior (MTP) > medium turbinate anterior (MTA). Uptake and transport also exhibited directionality and temperature dependence in these tissues. Further, uptake as well as transport of functionalized nanoparticles could be inhibited by excess free functionalizing ligand. Confocal microscopy indicated the presence of functionalized nanoparticles in respiratory epithelial cells, as well as other cell types of the nasal tissue. We chose the ITP region for further studies with deslorelin or transferrin-conjugated poly-l-lactide-co-glycolide nanoparticles (PLGA-NPs) encapsulating an anti-VEGF intraceptor (Flt23k) plasmid. Transport of the nanoparticles, as well as the plasmid from the nanoparticles, exhibited the following order: transferrin-PLGA-NPs > deslorelin-PLGA-NPs > PLGA-NPs ≫ plasmid. The ability of the nanoparticles transported across the nasal tissue to retain the effectiveness of the Flt23k plasmid was evaluated by measuring transfection efficiency (percentage of cells expressing GFP) and VEGF inhibition in LNCaP and PC-3 prostate cancer cells. Transfection efficiencies and VEGF inhibition in LNCaP and PC-3 cells exhibited the following trend: transferrin-PLGA-NPs ≥ deslorelin-PLGA-NPs > PLGA-NPs ≫ plasmid. Further, functionalized nanoparticles exhibited transfection efficiencies and VEGF inhibition significantly superior compared with the routinely used transfecting agent, lipofectamine. Formulating plasmids into nanoparticulate delivery systems enhances the transnasal delivery and gene therapy at remote target cancer cells, which can be further enhanced by nanoparticle functionalization with deslorelin or transferrin.—Sundaram, S., Roy, S. K., Ambati, B. K., Kompella, U. B. Surface functionalized nanoparticles for targeted gene delivery across nasal respiratory epithelium.

Estrogen receptors (ERs) [ERalpha (Esr1) and ERbeta (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ERalpha and ERbeta is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ERalpha knockout and Apc(Min) mouse strains, we demonstrate that ERalpha deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in Apc(Min/+) mice. Within the normal intestinal epithelium of Apc(Min/+) mice, ERalpha deficiency is associated with an accumulation of nuclear beta-catenin, an indicator of activation of the Wnt-beta-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ERalpha deficiency is associated with activation of Wnt-beta-catenin signaling, ERalpha deficiency in the intestinal epithelium of Apc(Min/+) mice also correlated with increased expression of Wnt-beta-catenin target genes. Through crosses between an ERbeta knockout and Apc(Min) mouse strains, we observed some evidence that ERbeta deficiency is associated with an increased incidence of colon tumors in Apc(Min/+) mice. This effect of ERbeta deficiency does not involve modulation of Wnt-beta-catenin signaling. Our studies suggest that ERalpha and ERbeta signaling modulate colorectal carcinogenesis, and ERalpha does so, at least in part, by regulating the activity of the Wnt-beta-catenin pathway.

FSH stimulates DNA synthesis by hamster preantral follicles both in vivo and in vitro, and the in vitro mitogenic effect of FSH is effectively reproduced by epidermal growth factor (EGF). To determine whether follicular EGF is the intracellular transducer of FSH action on hamster preantral follicles, intact follicles at stages 1 to 7 (stages 1-4 = preantral follicles 1-4 layers of granulosa cells, respectively, and no theca; stages 5-6 = 5-6 and 7-8 layers granulosa cells, respectively, and developing theca; and stage 7 = follicles with incipient antrum) were cultured for 24 h in a serum-free culture medium in the absence or presence of 100 ng FSH, 50 ng EGF, 50 ng transforming growth factor-alpha (TGF alpha), or 1 mumol 8-Br-cAMP and challenged with 50 microliters polyclonal antimurine EGF antiserum; the rate of DNA synthesis was determined by [3H]thymidine incorporation. FSH, EGF, and TGF alpha significantly (P less than 0.05) stimulated follicular DNA synthesis; TGF alpha per se was less effective than either FSH or EGF. However, both FSH- and EGF-induced DNA synthesis was drastically attenuated by EGF antiserum; TGF alpha effect remained undisturbed. Interestingly antibody inhibition of FSH-induced DNA synthesis was totally reversed by coexposure to TGF alpha. Follicular DNA synthesis for most stages was stimulated by Br-cAMP, but the effect was significantly (P less than 0.05) inhibited by EGF antibody. Moreover, follicles at different stages responded to EGF with different latency. These results strongly suggest that FSH-induced follicular DNA synthesis in the hamster is mediated by follicular EGF and the pathway of events is FSH action----cAMP production----EGF synthesis----cell proliferation.

Preantral (stages 1-6) and antral (stages 7-10) follicles from proestrous hamsters were exposed for 24 h to 1, 5, 10, 50, and 100 ng/ml of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and fibroblast growth factor (FGF) and 1 microCi [3H]thymidine to determine the rate of DNA replication. FSH (100 ng/ml) was used as a positive control. Synergism was also studied by using suboptimal doses of growth factors (1 ng/ml) and FSH (5 ng/ml). Optimal doses (50 ng/ml) of EGF, IGF-I, and FGF, and 100 ng/ml FSH significantly enhanced follicular DNA replication, whereas suboptimal doses of FSH, EGF, and IGF-I affected only a few preantral stages, and FGF was totally ineffective. FGF significantly inhibited DNA synthesis induced by a suboptimal dose of EGF but did not affect IGF-I-induced DNA replication. Paradoxically, a combination of suboptimal doses of all three growth factors significantly (p less than 0.05) stimulated DNA synthesis for all stages; FSH (5 ng/ml) had no additive effect. FSH significantly stimulated follicular progesterone (P4), androstenedione (A), and estradiol-17 beta (E2) accumulation, but significant P4 accumulation from stages 3-10 was observed only after optimal EGF exposure; A and E2 accumulations were unaffected by any of the growth factors. These results indicate that EGF, IGF-I, and FGF stimulate DNA replication to hamster preantral and antral follicles, and may play roles as intraovarian factors regulating folliculogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

In recent studies, we have shown that the smallest preantral follicles in the cyclic hamster increase DNA synthesis in the periovulatory period in response to surge levels of FSH. The current investigation was designed to determine whether the same phenomenon occurs in the cyclic mouse. Intact mouse follicles were isolated with watchmaker forceps (stages 4-6) or by enzymatic digestion (stages 1-4) at 0900 h and 1500 h on each day of the 5-day estrous cycle. The isolated follicles were classified into 6 stages: stages 1 and 2: follicles with 1 and 2 layers of granulosa cells; stage 3: follicles with 3 or more layers of granulosa cells and formation of theca; stages 4-6: incipient, small, and preovulatory antral follicles. The follicles at each stage were incubated for 3 h with [3H]thymidine. DNA content in stages 1-4 of follicles remained unchanged during the estrous cycle; for stages 5 and 6, DNA content was higher on the afternoon of proestrus than on other days of the cycle. Incorporation of [3H]thymidine for stages 1-3 (preantral follicles) started to increase at 1500 h of proestrus and peaked at 0900 h on estrus, whereas for stages 4-6, DNA synthesis peaked on proestrus (1500 h) and then fell by the morning of estrus. Thus, the rate of DNA replication in preantral and antral mouse follicles were different. Similarities and differences in folliculogenesis between mouse and hamster are discussed. These results suggest that DNA synthesis and the growth of all stages of follicles in the cyclic mouse may be associated with changing levels of periovulatory gonadotropins.

Deslorelin, a luteinizing hormone releasing hormone (LHRH) agonist, is transported via the LHRH-receptor (LHRH-R) and exhibits regional variation as follows: inferior turbinate posterior (ITP) > medium turbinate posterior (MTP) > medium turbinate anterior (MTA) of the bovine nasal mucosa. Differential LHRH-R expression in various regions of the nose is a potential explanation for regional variation in deslorelin transport. Thus, the objective was to determine whether LHRH-R expression exhibits regional variation in bovine nasal mucosa. LHRH-R density ( B max ) and affinity constant ( K d ) were determined by saturation experiments using 0.5 mg tissue in the presence of increasing amounts of I 125 -deslorelin (100–100,000 cpm) at 4 °C for 4 h. The 50% inhibitory concentration (IC 50 ) was determined by competition experiments using various amounts of unlabelled deslorelin (0.01–3000 ng) at 4 °C for 4 h. LHRH-R mRNA and protein expressions were determined using real-time PCR and Western blot analysis, respectively. LHRH-R B max and K d varied between the regions of excised bovine nasal mucosa: ITP > MTP > MTA. The inhibition experiments yielded two IC 50 concentrations which exhibited trends similar to B max and K d . Real-time PCR and Western blot analysis indicated that LHRH-R expression exhibits similar trends: ITP > MTP > MTA. We identified two deslorelin binding sites in the nasal tissues, with high affinity sites representing approximately 60–70% of the total sites available. In summary, regional differences in nasal deslorelin transport correlate with regional differences in LHRH-R expression, with LHRH-R expression, peptide binding, and transport being the highest in the inferior turbinate posterior region of the nose.

Estradiol-17 (E2) plays an important role in the formation and development of primordial follicles, but the mechanisms remain unclear. G protein-coupled receptor 30 (GPR30) can mediate a rapid and transcription-independent E2 signaling in various cells. The objectives of this study were to examine whether GPR30 was expressed in the neonatal hamster ovary and whether it could mediate estrogen action during the formation of primordial follicles. GPR30 mRNA levels decreased from the 13th day of gestation (E13) through the second day of postnatal (P2) life, followed by steady increases from P3 through P6. Consistent with the changes in mRNA levels, GPR30 protein expression decreased from E13 to P2 followed by a significant increase by P7, the day before the first appearance of primordial follicles in the hamster ovary. GPR30 was expressed both in the oocytes and somatic cells, although the expression in the oocytes was low. GPR30 protein was located primarily in the perinuclear endoplasmic reticulum, which was also the site of E2-BSA-FITC (E2-BSA-fluorescein isothiocyanate) binding. E2 or E2-BSA increased intracellular calcium in neonatal hamster ovary cells in vitro. Exposure to GPR30 small interfering RNA in vitro significantly reduced GPR30 mRNA and protein levels in cultured hamster ovaries, attenuated E-BSA binding to cultured P6 ovarian cells, and markedly suppressed estrogen-stimulated primordial follicle formation. These results suggest that a membrane estrogen receptor, GPR30, is expressed in the ovary during perinatal development and mediates E2 action on primordial follicle formation. (Endocrinology 149: 4452–4461, 2008)