Human hair pigmentation--biological aspects

Skin and hair colour contribute significantly to our overall visual appearance and to social/sexual communication. Despite their shared origins in the embryologic neural crest, the hair follicle and epidermal pigmentary units occupy distinct, although open, cutaneous compartments. They can be distinguished principally on the basis of the former's stringent coupling to the hair growth cycle compared with the latter's continuous melanogenesis. The biosynthesis of melanin and its subsequent transfer from melanocyte to hair bulb keratinocytes depend on the availability of melanin precursors and on a raft of signal transduction pathways that are both highly complex and commonly redundant. These signalling pathways can be both dependent and independent of receptors, act through auto-, para- or intracrine mechanisms and can be modified by hormonal signals. Despite many shared features, follicular melanocytes appear to be more sensitive than epidermal melanocytes to ageing influences. This can be seen most dramatically in hair greying/canities and this is likely to reflect significant differences in the epidermal and follicular microenvironments. The hair follicle pigmentary unit may also serve as an important environmental sensor, whereby hair pigment contributes to the rapid excretion of heavy metals, chemicals and toxins from the body by their selective binding to melanin; rendering the hair fibre a useful barometer of exposures. The recent availability of advanced cell culture methodologies for isolated hair follicle melanocytes and for intact anagen hair follicle organ culture should provide the research tools necessary to elucidate the regulatory mechanisms of hair follicle pigmentation. In the longer term, it may be feasible to develop hair colour modifiers of a biological nature to accompany those based on chemicals.     

Versican expression by dermal papilla-regenerated hair follicles – a promising tool for hair-regrowth products

Hair follicle stem cells and dermal papilla cells play essential roles in the cyclical hair regeneration process. However, compared with the recent progress of follicular stem cell research, detailed analysis of dermal papilla cells is virtually unknown. We have focused on a large chondroitin sulfate proteoglycan molecule, versican, and prove its pivotal role in hair follicle induction both in vitro and in vivo. First, we examined versican expression with hair cycling by in situ hybridization (mRNA) and immunohistochemistry (protein). Results clearly showed specific versican expression in anagen onset of dermal papilla cells implying a function role of versican in hair induction. We then generated transgenic lines with LacZ or green fluorescent protein reporter genes under a versican regulatory element (promoter) control, and confirmed dermal papilla‐specific activation of the reporter. These transgenic lines were utilized to isolate the dermal papilla cell population by means of a fluorescent activated cell sorter. While freshly isolated dermal papilla cells were able to induce hair growth in a skin reconstitution assay when grafted with undifferentiated epidermal cells, this ability and versican expression were rapidly lost during passages in culture. In order to examine the functional role of versican in hair growth induction, versican full‐length cDNAs were transfected into inactive passaged dermal papilla cells. Impressively, this forced expression of versican molecules partially restored hair inductivity of dermal papilla cells in a skin reconstitution assay. These results demonstrated the pivotal role of versican in hair induction both in vitro and in vivo and will connect the missing link between the signal from dermal papilla cells to follicular stem cells, and initiation of hair regeneration. We have also shown that the hair growth‐promoting compound, cyclosporin A, can upregulate versican expression implying the application of a novel screening approach for hair growth‐promoting compounds by monitoring versican expression as an indicator.     

The regulatory role of the tetrapeptide AcSDKP in skin and hair physiology and the prevention of ageing effects in these tissues – a potential cosmetic role

The naturally occurring tetrapeptide acetyl‐N‐Ser‐Asp‐Lys‐Pro (AcSDKP) recognized as a potent angiogenic factor was shown recently to contribute to the repair of cutaneous injuries. In the current article, we report the ability of AcSDKP to exert a beneficial effect on normal healthy skin and scalp and to compensate for the ageing process. In vitro AcSDKP at 10^?11–10^?7 M significantly stimulates the growth of human keratinocytes, fibroblasts and follicle dermal papilla cells. Moreover, it enhances the growth of human epidermal keratinocyte progenitor and stem cells as shown in a clonogenic survival assay. Topical treatment of ex vivo cultured skin explants with 10^?5 M AcSDKP increases the thickness of the epidermis and upregulates the synthesis of keratins 14 and 19, fibronectin, collagen III and IV as well as the glycoaminoglycans (GAGs). In the ex vivo‐cultured hair follicles, AcSDKP promotes hair shaft elongation and induces morphological and molecular modifications matching the criteria of hair growth. Furthermore, AcSDKP at 10^?11–10^?7 M was shown to improve epidermal barrier, stimulating expression of three protein components of tight junctions (claudin‐1, occludin, ZO‐1) playing an important role in connecting neighbouring cells. This tetrapeptide exercises also activation of SIRT1 implicated in the control of cell longevity. Indeed, a two‐fold increase in the synthesis of SIRT1 by cultured keratinocytes was observed in the presence of 10^?11–10^?7 M AcSDKP. In conclusion, these findings provide convincing evidence of the regulatory role of AcSDKP in skin and hair physiology and suggest a cosmetic use of this natural tetrapeptide to prevent skin ageing and hair loss and to promote the cutaneous regeneration and hair growth.     

Luteinization of porcine preovulatory follicles leads to systematic changes in follicular gene expression

The LH surge initiates the luteinization of preovulatory follicles and causes hormonal and structural changes that ultimately lead to ovulation and the formation of corpora lutea. The objective of the study was to examine gene expression in ovarian follicles (n = 11) collected from pigs (Sus scrofa domestica) approaching estrus (estrogenic preovulatory follicle; n = 6 follicles from two sows) and in ovarian follicles collected from pigs on the second day of estrus (preovulatory follicles that were luteinized but had not ovulated; n = 5 follicles from two sows). The follicular status within each follicle was confirmed by follicular fluid analyses of estradiol and progesterone ratios. Microarrays were made from expressed sequence tags that were isolated from cDNA libraries of porcine ovary. Gene expression was measured by hybridization of fluorescently labeled cDNA (preovulatory estrogenic or -luteinized) to the microarray. Microarray analyses detected 107 and 43 genes whose expression was decreased or increased (respectively) during the transition from preovulatory estrogenic to -luteinized (P<0.01). Cells within preovulatory estrogenic follicles had a gene-expression profile of proliferative and metabolically active cells that were responding to oxidative stress. Cells within preovulatory luteinized follicles had a gene-expression profile of nonproliferative and migratory cells with angiogenic properties. Approximately, 40% of the discovered genes had unknown function.     

Gonadotrophin responsiveness, aromatase activity and insulin-like growth factor binding protein content of bovine ovarian follicles during the first follicular wave

The aim of this study was to examine the function of granulosa cells and hormone concentrations in follicular fluid in bovine ovarian follicles during selection of the first dominant follicle. Ovaries were obtained from beef heifers on days 1-5 after ovulation: follicles > 4 mm in diameter were dissected and follicular fluid and granulosa cells were collected from individual follicles. Oestradiol production by granulosa cells after culture with testosterone was used to determine aromatase activity and responsiveness to gonadotrophins was determined by cAMP production after culture with FSH or LH. Concentrations of oestradiol, progesterone and insulin-like growth factor binding proteins (IGFBPs)-4 and -5 were measured in follicular fluid. Follicles were classified as largest or smaller (days 1 and 2), or dominant or subordinate (days 3-5). Aromatase activity was greater in granulosa cells from the largest follicle than in granulosa cells from smaller follicles on days 1, 3, 4 and 5 (P < 0.05). Responsiveness to LH was not detected in granulosa cells on day 1, but from day 2 to day 5 cells from the largest follicle were significantly more responsive than cells from smaller follicles (P < 0.05). Responsiveness to FSH was detected in granulosa cells from all follicles from day 1 onwards and did not differ between cells from the largest follicle or smaller follicles on any day. Follicular fluid concentrations of oestradiol and the ratio of oestradiol:progesterone were greater and concentrations of IGFBP-4 and -5 were lower in the largest follicle than in smaller follicles from day 2 to day 5 (P < 0.05). In conclusion, selection of the dominant follicle is associated with increased granulosa cell aromatase activity followed by increased cAMP response to LH and follicular fluid oestradiol concentrations, and decreased follicular fluid concentrations of IGFBP-4 and -5 within 2 days after ovulation.     

The biology of hair diversity

Hair diversity, its style, colour, shape and growth pattern is one of our most defining characteristics. The natural versus temporary style is influenced by what happens to our hair during our lifetime, such as genetic hair loss, sudden hair shedding, greying and pathological hair loss in the various forms of alopecia because of genetics, illness or medication. Despite the size and global value of the hair care market, our knowledge of what controls the innate and within‐lifetime characteristics of hair diversity remains poorly understood. In the last decade, drivers of knowledge have moved into the arena of genetics where hair traits are obvious and measurable and genetic polymorphisms are being found that raise valuable questions about the biology of hair growth. The recent discovery that the gene for trichohyalin contributes to hair shape comes as no surprise to the hair biologists who have believed for 100 years that hair shape is linked to the structure and function of the inner root sheath. Further conundrums awaiting elucidation include the polymorphisms in the androgen receptor (AR) described in male pattern alopecia whose location on the X chromosome places this genetic contributor into the female line. The genetics of female hair loss is less clear with polymorphisms in the AR not associated with female pattern hair loss. Lifestyle choices are also implicated in hair diversity. Greying, which also has a strong genetic component, is often suggested to have a lifestyle (stress) influence and hair follicle melanocytes show declining antioxidant protection with age and lowered resistance to stress. It is likely that hair research will undergo a renaissance on the back of the rising information from genetic studies as well as the latest contributions from the field of epigenetics.     

Coenzyme Q10 has anti-aging effects on human hair

IFSCC Magazine, 11 (2008) (1) 37–42 Ubiquinones are the most widespread and therefore best investigated bioquinones. Due to their hydrophobic isoprenoid side chain, ubiquinones can be solubilized in organic solvents or lipids but are insoluble in water. Using a specific emulsifier system it has been possible to deliver positive effects to biological systems also from aqueous formulations. Ubiquinone‐50, also referred to as coenzyme Q10, is well known in cosmetic science and especially in skin care because of its antioxidant activity. It is found in the membranes of peroxisomes, lysosomes, vesicles, the endoplasmic reticulum and notably in the inner membrane of the mitochondrion, where it is an important part of the electron transport chain. Using coenzyme Q10 in the proposed emulsifier system we could show that the molecule not only has relevance as an anti‐aging bioactive in skin care but also has positive effects on the human hair follicle. The hair follicle is a complex mini organ and synthesis of hair keratin, the major component of hair fibers, is an essential prerequisite for the growth of strong and healthy hair. But like all biological systems the hair follicle, the biologically active part of the hair, also undergoes an aging process associated among other things with a decline in certain hair keratins. Due to this age‐related shift in basic structural proteins of the hair shaft, mature hair often becomes fragile and difficult to manage. Therefore it is a challenge for cosmetic science to provide bioactives to fight age‐related changes and maintain a youthful appearance of hair. Using cultivated hair follicle keratinocytes we identified coenzyme Q10 as a potent bioactive that stimulates the gene expression of different hair keratins, especially those which are reduced during aging processes in hair follicles. These results led us to investigate a shampoo and a tonic formulation enriched with coenzyme Q10 in a placebo‐controlled panel study. In a left/right comparison a group of healthy volunteers older than 40 years of age applied the formulations daily for 4 days. Throughout the test period the gene expression of different hair keratins from plucked hair follicles was determined using quantitative polymerase chain reaction techniques. Subsequent statistical analysis revealed an increase in age‐relevant hair keratins in human hair roots treated with coenzyme Q10, thus pointing out the striking benefits of coenzyme Q10 in hair care formulations. We conclude that coenzyme Q10 is an ideal ingredient for hair care formulations, providing anti‐aging properties through activation of specific keratins aligned with the needs of mature hair.     

The primordial follicle reserve is not renewed after chemical or γ-irradiation mediated depletion

Reports indicate that germ-line stem cells present in adult mice can rapidly generate new oocytes and contribute to the primordial follicle reserve following conditions of ovotoxic stress. We further investigated the hypothesis that adult mice have the capacity to generate new oocytes by monitoring primordial follicle numbers throughout postnatal life and following depletion of the primordial follicle reserve by exposure to doxorubicin (DXR), trichostatin A (TSA), or whole-body γ-irradiation. We show that primordial follicle number remains stable in adult C57BL/6 mice between the ages of 25 and 100 days. However, within 2 days of treatment with DXR or TSA, primordial follicle numbers had declined to 65 and 51% respectively (P<0.05-0.01 when compared to untreated controls), with no restoration of follicle numbers evident after 7 days for either treatment. Furthermore, ovaries from mice subjected to sterilizing doses of γ-irradiation (0.45 or 4.5 Gy) revealed complete ablation of all primordial follicles 5 days after treatment, with no indication of follicular renewal. We conclude that neo-folliculogenesis does not occur following chemical or γ-irradiation mediated depletion of the primordial follicle reserve.     

Grey hair: clinical investigation into changes in hair fibres with loss of pigmentation in a photoprotected population

Loss of pigmentation in hair fibres is one of the most obvious phenotypic changes with ageing and has been a topic of increasing interest in the study of follicle biology. The onset of greying brings cosmetic complaints that grey fibres are wild or difficult to manage. Of course, these perceptions may be the consequence of visual obviousness rather than underlying physical or chemical differences. Although several studies have compared pigmented and unpigmented fibres, few have tried to control genetic and ethnic difference as well as extrinsic factors such as photoexposure and chemical treatment. We have recruited subjects with salt-and-pepper hair from a population of Old Order Mennonites who, for cultural reasons, are not only prohibited from chemically treating their hair but also limit their exposure to sunlight. Hair samples were examined for elemental composition, surface energy, Young's modulus, break stress, bending modulus, shear modulus and water sorption/desorption isotherm. The parameters were evaluated statistically for global differences, individual differences and typical individual differences. Consistent with previous published literature, few global differences were found between pigmented and unpigmented hair across the population. We do find that many individual subjects had differences between pigmented and unpigmented fibres. These differences tend to be more pronounced in bulk than in surface properties. The small differences in mechanical properties and moisture uptake and loss lend support to the perception by consumers that grey hair is wilder, drier and less manageable.     

BAX is involved in regulating follicular growth, but is dispensable for follicle atresia in adult mouse ovaries

Mammalian females are endowed with a finite number of primordial follicles at birth or shortly thereafter. Immediately following the formation of the primordial follicle pool, cohorts of these follicles are recruited to begin growth, and this recruitment continues until the primordial follicle population is depleted. Once recruited, a follicle will either grow and ovulate or undergo atresia. Follicle atresia results from the apoptotic death of follicular cells. Members of the BCL-2 family of proteins are important regulators of apoptosis in most cells including in the ovary. Here, we tested the hypothesis that the proapoptotic BAX is an important regulator of follicle survival. We used a variety of histological and biochemical techniques to investigate the impact of Bax deletion on follicle growth and death. We observed that the Bax deletion results in delayed vaginal opening and altered follicular growth. Young adult Bax-deficient ovaries contained increased numbers of primordial follicles and a trend towards reduced numbers of growing follicles. Bax deficiency led to a reduction in average litter size, and also a reduction in the number of oocytes ovulated in response to exogenous gonadotropins. In contrast, Bax deficiency did not alter follicle atresia. In conclusion, BAX appears to be an important regulator of follicle growth, but is dispensable for follicle atresia in mice.     

Composition and morphology of the follicular basal lamina during atresia of bovine antral follicles

The fate of the follicular basal lamina during atresia was investigated using bovine follicles, in which different follicle phenotypes have been observed. These phenotypes include: healthy follicles with rounded basal granulosa cells with an aligned basal lamina or follicles with columnar basal granulosa cells with a basal lamina of many loops (loopy), and atretic follicles in which either the antral granulosa cells (antral atresia) or the basal cells (basal atresia) die first. Loopy lamina and basal atresia occur only in small antral follicles < 5 mm in diameter. Follicles were collected from cattle of unknown reproductive history and processed for immunohistochemistry and electron microscopy, and from animals in which follicle growth had been monitored by daily measurements of follicle diameter by ultrasonography. Electron microscopic observations of dominant follicles during the growth phase, plateau and regression showed that the basal lamina was still visible and intact upon atresia. These follicles had a conventional aligned basal lamina, which they retained, except for some degree of folding, as they progressed into antral atresia. In small follicles (2-5 mm in diameter), the basal cell shape (rounded or columnar) and appearance of the basal lamina (aligned or of many loops) did not appear to be related to the type of atresia. On atresia the follicular basal laminae retained immunoreactive laminin alpha1 and beta2, type IV collagen alpha1 and nidogen. Laminin alpha2, which may come from the theca, was present in the follicular basal lamina of only 22% of healthy follicles, but was expressed very commonly in 71% of the atretic follicles. Laminin alpha2 expression was found in both phenotypes of healthy follicles, antral and basal atretic follicles, and follicles with aligned or loopy basal laminae. It is concluded that the basal lamina is not degraded upon atresia, but does undergo a variety of other changes.     

Akt and Erk signal transduction pathways are early markers of differentiation in dominant and subordinate ovarian follicles in cattle

Dominant follicles are those that continue to develop and have the potential to ovulate while subordinate follicles regress. Characteristics of dominant follicles include a larger diameter, higher intrafollicular estradiol, and lower IGF-binding protein (IGFBP)-4 concentrations compared with other cohort follicles. Follicle development is regulated by endocrine hormones that act via intracellular signaling pathways. Here, we show the differences in Akt, Erk, c-Jun N-terminal protein kinase, and p-38 signaling pathways between dominant and subordinate follicles at the dominance stage of the follicle wave. However, earlier in the follicle wave (dominant follicle selection), there were only differences in the levels of Akt and Erk signal transduction proteins among dominant and subordinate follicles. Using this profile of Akt and Erk protein expression in granulosa and theca cells of selected dominant follicles compared with subordinate follicles, we suggest a predictive model to identify future dominant and subordinate follicles from the pool of otherwise similar cohort follicles at the time of follicle wave emergence. We conclude that the Erk and Akt signal transduction pathways are important for dominant follicle selection and development and, furthermore, that the observed differences in these pathways mark the future dominant follicle from subordinate follicles before differences in follicular diameter, follicular fluid estradiol, and IGFBP-4 concentrations are apparent.     

Regulation of apoptosis in the atresia of dominant bovine follicles of the first follicular wave following ovulation

During atresia of bovine follicles, granulosa cells are lost through the controlled form of cell death, apoptosis. The purpose of this study was to characterize the regulation of apoptotic death of granulosa cells in dominant bovine follicles during the first wave of follicular development. Dominant follicles were collected from Holstein heifers on days 4, 6 or 8 of the first follicular wave (n = 5/day). Regulation of apoptosis in granulosa cells was examined by annexin V and propidium iodide staining; measurement of relative levels of mRNA encoding Bcl-2, Bcl-xL and Bax; and activity of caspase-3, -8 and -9. Steady-state levels of mRNA encoding four oxidative stress-response proteins were determined. Compared with day 4, the incidence of apoptotic and nonviable granulosa cells tended to increase on day 6, and numbers of nonviable cells were higher on day 8. The ratios of relative levels of mRNA encoding Bcl-2 to Bax and Bcl-xL to Bax were higher on day 6 than days 4 and 8. Activity of caspases-3 and -9 in granulosa cells did not change among the 3 days, while caspase-8 activity decreased on day 8 compared with days 4 and 6. Amounts of GSHPx, MnSOD and Cu/ZnSOD mRNA in granulosa cells were higher on day 8 than day 6. In theca interna, amounts of Cu/ZnSOD mRNA decreased between days 4 and 6. From the decreased production of estradiol and increased numbers of apoptotic and nonviable granulosa cells, we conclude that atresia of the dominant follicle is initiated between days 4 and 6 of the first follicular wave. However, apoptosis of granulosa cells does not appear to be initiated by changes in expression of oxidative stress-response proteins.     

Heat stress diminishes gonadotropin receptor expression and enhances susceptibility to apoptosis of rat granulosa cells

Heat stress inhibits ovarian follicular development in mammalian species. We hypothesized that heat stress inhibits the function of follicular granulosa cells and suppresses follicular development. To test this, immature female rats were injected with pregnant mare serum gonadotropin (PMSG) at 48 h after the start of temperature treatment (control: 25 degrees C, 50% RH; heat stress: 35 degrees C, 70% Relative Humidity). The ovaries and granulosa cells of follicles at different developmental stages were analyzed for gonadotropin receptor levels and aromatase activity; estradiol levels were measured in follicular fluid. Before injection, heat stress diminished only the amount of FSH receptor on granulosa cells of antral follicles. During PMSG-stimulated follicular development, heat stress strongly inhibited gonadotropin receptor levels and aromatase activity in granulosa cells, and estradiol levels in the follicular fluid of early antral, antral and preovulatory follicles. To examine apoptosis and mRNA levels of bcl-2 and bax in granulosa cells, follicles harvested 48 h after PMSG injection were cultured in serum-free conditions. Heat-stressed granulosa cells showed a time-dependent increase in apoptosis. The bcl-2 mRNA levels were similar in control and heat-stressed granulosa cells; bax mRNA levels were increased in heat-stressed granulosa cells. According to these results, heat stress inhibits expression of gonadotropin receptors in granulosa cells and attenuates estrogenic activity of growing follicles, granulosa cells of heat-stressed follicles are susceptible to apoptosis, and the bcl2/bax system is not associated with heat-stress-induced apoptosis of granulosa cells. Our study suggests that decreased numbers and function of granulosa cells may cause ovarian dysfunction in domestic animals in summer.     

Alterations in follicular dynamics and steroidogenic abilities induced by heat stress during follicular recruitment in goats

We investigated the changes in follicular dynamics and steroidogenic activity during heat stress in goats. Adult female goats were exposed to heat stress at 36 degrees C and 70% relative humidity for 48 h and then injected with prostaglandin (PG) F2alpha (the time of PGF2alpha injection was designated as 0 h). In experiment 1, every follicle greater than 2 mm in diameter was monitored by ultrasonography to investigate the follicular dynamics, and plasma concentrations of FSH, LH, progesterone, and oestradiol were measured from -48 h to 120 h. In experiment 2, the follicles were recovered from the goats at 48 h, and the concentration of oestradiol, the aromatase activity, and the LH receptor level in the follicles were determined. In control (non-heat-stressed) goats, ovulatory follicles were mainly recruited from -24 h to 0 h, whereas no follicles recruited during that period were ovulated in the heat-stressed goats. The timing of the recruitment of ovulatory follicles was delayed by heat stress by approximately 24 h. The plasma concentration of oestradiol in the heat-stressed goats was significantly lower from 36 to 54 h compared with the controls, although the concentrations of FSH and progesterone did not differ between the treatments. In addition, the concentration of oestradiol, the aromatase activity, and the LH receptor level in the follicles from heat-stressed goats were significantly lower compared with the controls. These results indicate that heat stress during follicular recruitment suppresses subsequent growth to ovulation, accompanied by decreased LH receptor level and oestradiol synthesis activity in the follicles.     

多不饱和脂肪酸干预脱发症的研究现状及作用机制

脱发症是皮肤科常见疾病之一,发病率较高但临床治疗手段有限。大部分脱发症与毛囊周期的改变有关,而Omega-3及Omega-6多不饱和脂肪酸被证实通过促进或抑制毛囊细胞释放信号分子,从而促进毛囊生长、改善毛囊周期以及促进毛囊的再生。对多不饱和脂肪酸干预脱发症的研究现状及作用机制的国内外相关研究进行综述,期望能够为多不饱和脂肪酸对各类脱发症的治疗提供新思路及深入探讨脱发症的治疗方法及治疗机制提供借鉴,为利用现代分子生物技术改善脱发症提供理论依据。     

Development and validation of a short-term, serum-free culture system for bovine granulosa cells: evaluation of the effects of somatotropin and growth hormone-releasing factor on estradiol production

The objective of this study was to develop and validate a short-term, serum-free culture system to determine whether recombinant bovine somatotropin (rbST) or recombinant bovine growth hormone-releasing factor (rbGRF) altered the estradiol-producing capacity of bovine granulosa cells isolated from dominant or subordinate follicles of the first follicular wave. Thus, ovaries were obtained at an abattoir from cows that were between d 2 to 5 or 6 to 10 of the estrous cycle. Three size classes of follicles were isolated from each cow's ovaries: small (2 to 5 mm in diameter), medium (6 to 14 mm), or the largest (6 to 19 mm). In vivo steroid-producing capacity of follicles was assessed by measuring concentration of estradiol, progesterone, androstenedione and 5alpha-dihydrotestosterone in each follicle. In vitro steroid-producing capacity was assessed by culturing granulosa cells from the different follicle sizes for 48 h in serum-free media with 19-OH androstenedione and measuring the estradiol and progesterone concentrations in media at the end of culture. The effect of different doses of FSH, rbST, or rbGRF on estradiol and progesterone production by granulosa cells from each follicle size class during d 2 to 5 or 6 to 10 was also evaluated. A high percentage (91.7%) of the largest follicles obtained on d 2 to 5 was estrogen-active (estradiol > progesterone) compared with other follicle classifications (d 2 to 5, small = 0%, medium = 13.8%; d 6 to 10, small = 0%, medium = 3.3%, largest = 33.3%). Estradiol was highest (P < 0.05) in the largest follicle on d 2 to 5 and correlated positively with follicle diameter. The pattern of in vitro production of estradiol by granulosa cells from the different follicle size classes reflected the original in vivo capacity of follicles to produce estradiol. However, only granulosa cells from the largest estrogen-active follicle on d 2 to 5 produced more estradiol than progesterone in vitro. Progesterone production by granulosa cells from all follicle classifications was increased by FSH, but FSH only enhanced estradiol production by granulosa cells from the largest estrogen-active follicles on d 2 to 5. Recombinant bST blocked the FSH-induced increase in estradiol by granulosa cells from the largest estrogen-active follicles on d 2 to 5, whereas rbGRF had no effect on steroid production. Based on these results, we concluded that short-term, serum-free culture of bovine granulosal cells obtained from first-wave follicles at an abattoir could be used to reflect reliably the original in vivo estradiol-producing capacity of granulosal cells, and that neither rbST nor rbGRF enhance basal or FSH-induced estradiol production by bovine granulosa cells from first-wave follicles.     

Curcumin induces heme oxygenase-1 in normal human skin fibroblasts through redox signaling: relevance for anti-aging intervention

Scope: Curcumin, a component of the spice turmeric, was tested for its potential hormetic anti‐aging effects as an inducer of mild stress. Methods and results: Early passage young human skin fibroblasts treated with low doses of curcumin (below 20 μM) showed a time‐ and concentration‐dependent induction of heme oxygenase‐1 (HO‐1), followed by compensatory increase in glutathione‐S‐transferase activity, GSH levels and GSH/GSSG ratio. These effects were preceded by induction of oxidative stress (increased levels of reactive oxygen species and DNA damage) and impairment of cells' GSH redox state. Curcumin also induced nuclear factor‐erythroid‐2‐related factor 2 accumulation in the nuclei. The use of the antioxidant N‐acetyl cysteine prevented the induction of HO‐1 by curcumin. Pharmacological inhibition of phosphatidylinositol 3‐kinase, but not other kinases, significantly prevented curcumin‐induced HO‐1 levels, which was corroborated by the induction of phospho‐Akt levels by curcumin. Late passage senescent cells already had higher HO‐1 levels, and further induction of HO‐1 by curcumin was considerably impaired. The induction of stress responses by curcumin in human cells led to protective hormetic effects to further oxidant challenge. Conclusion: Curcumin induces cellular stress responses in normal human skin fibroblasts through phosphatidylinositol 3‐kinase/Akt pathway and redox signaling, supporting the view that curcumin‐induced hormetic stimulation of cellular antioxidant defenses can be a useful approach toward anti‐aging intervention.     

Effects of ovarian follicle ablation on FSH, oestradiol and inhibin A concentrations and growth of other follicles in sheep

The aim of this study was to examine the effect of removal of the largest follicle or all visible follicles during the first follicle wave on subsequent follicular growth, steroid, inhibin A and gonadotrophin secretion in sheep. On day 4.5 of a synchronized oestrous cycle, ewes (n = 18) were assigned to one of three groups which underwent either no treatment (control), ablation of the largest follicle (largest follicle aspirated and cauterized via laparotomy) or ablation of all follicles (all visible follicles ablated). Between day 0 and day 10 of the oestrous cycle, blood samples were collected every 8 h and ovaries were examined daily using transrectal ultrasonography. The lifespan of the second largest follicle (number of days > 3 mm in diameter) was longer (6.7 +/- 0.9 days; P < 0.05) and the maximum diameter tended to be greater (4.8 +/- 0.3 mm; P = 0.07) in ewes in which the largest follicle was ablated than in the control ewes (3.8 +/- 0.4 days; 4.2 +/- 0.3 mm). There was no difference in the day of emergence of the second follicular wave between groups (day 6.9 +/- 0.4). However, the peak of the transient increase in FSH concentrations after ablation was earlier (day 5.67 +/- 0.15; P < 0.05) in ewes in which all follicles were ablated than in control ewes (day 6.72 +/- 0.36); the timing in ewes that had only the largest follicle ablated was intermediate (day 6.11 +/- 0.28). Serum inhibin A concentrations were about three-fold lower (P < 0.05) in both follicle ablation groups than in the control group. The numbers of follicles 2-3 mm in diameter during the first 3 days of the second follicular wave were greater in 'ablated ewes' (both groups had 2.6 +/- 0.2 follicles day-1) than in control ewes (1.7 +/- 0.3 follicles day-1). It is concluded that: (i) transient increases in FSH concentrations precede the emergence of follicle waves; (ii) ablation of all follicles on day 4.5 after oestrus advanced the timing of the next peak in FSH concentrations and the numbers of small follicles associated with the development of the second follicular wave; and (iii) ablation of the largest follicle resulted in an increase in the lifespan of the second largest follicle, indicating a regulatory role of large dominant follicles over smaller subordinate follicles.     

Short-term transplantation of isolated human ovarian follicles and cortical tissue into nude mice

This study was designed to evaluate follicular survival and growth after short-term transplantation of fresh isolated human follicles and ovarian cortical tissue to nude mice. Ovarian biopsies were obtained from nine women undergoing laparoscopy. Twelve nude mice were xenografted with an ovarian cortical fragment in the right ovarian bursa, and a clot containing isolated follicles in the left, for a period of 7 days. One ungrafted fragment was used as a control. Histological sections were analyzed to determine follicle number and stage. The proliferative status of follicular cells was assessed by Ki-67 immunostaining. A total of 659 follicles was analyzed by histology and 545 follicles by immunohistochemistry. The percentage of primordial follicles was found to be markedly reduced 1 week post-grafting when compared with ungrafted tissue, while the percentage of primary follicles had significantly increased. Only 8% of follicles showed Ki-67-positive granulosa cells before grafting, whereas 1 week after grafting, 71% of follicles in fragments and 67% of isolated follicles were Ki-67-positive (P<0.001). Moreover, the histological aspect of isolated follicle grafts was similar to that of grafted fragments: follicles were surrounded by vimentin-positive stroma-like tissue of human origin, as confirmed by fluorescent in situ hybridization with human-specific probes. Our results demonstrate, for the first time, that isolated human follicles are able to survive and grow after xenografting. This study also shows massive in vivo follicular activation after transplantation of grafted fragments and isolated follicles. One week after grafting, well-structured stroma-like tissue of human origin was observed around the isolated follicles. The potential origin of this stroma is discussed.