Transcriptional Networks Controlling Chondrocyte Proliferation And Differentiation In Endochondral Ossification

Me: This will be a very quick note for the reader on something else to take into account when analyzing how the change between the chondrocyte proliferation and differentiation zones in the growth plates happens. The steps are tightly regulated by growth factors which activate chondrocyte specific transcription factors. They are Sox9, Gli2/3, and Runx2.
Pediatric Nephrology, April 2010, Volume 25, Issue 4, pp 625-631

Transcriptional networks controlling chondrocyte proliferation and differentiation during endochondral ossification

  • Manuela Wuelling, Andrea Vortkamp
Abstract

During endochondral ossification bones are formed as cartilage templates in which chondrocytes proliferate, differentiate into hypertrophic chondrocytes and are gradually replaced by bone. Postnatally, remnants of embryonic chondrocytes remain in a restricted domain between the ossified regions of the bones forming the growth plate. The coordinated proliferation and differentiation of chondrocytes ensures the continuous elongation of the epiphyseal growth plates. The sequential changes between proliferation and differentiation are tightly regulated by secreted growth factors, which activate chondrocyte-specific transcription factors. Transcription factors that play critical roles in regulating cell-type-specific gene expression include Sox9, Gli2/3, and Runx2. The interaction of these transcription factors with general transcriptional regulators like histone-modifying enzymes provides an additional level of regulation to fine-tune the expression of target genes in different chondrocyte populations. This review will outline recent advances in the analysis of the complex transcriptional network that regulates the distinct steps of chondrocyte differentiation.

The Role Of Leptin In Endochondral Ossification

Me: This is one of those posts that is more for information and to help the reader (and me) to better understand the various endocine and molecular biological pathways and functions. From the two studies below, we see that leptin is needed for this list of functions

  • 1. increase in femur and humerus length
  • 2. decrease in length of the calcified zone hypertrophic zone relative to the entire hypertrophic zone. (which is a good thing)
  • 3. increased organized collagen fibril arrangement
  • 4. modulates several events associated with terminal differentiation of chondrocytes
  • 5. altered type X collagen mRNA expression (Type X Collagen is produced by the chondrocytes in the hypertropic layer)
  • 6. suppressed apoptosis, cell growth and matrix calcification
  • 7. acts on human marrow stromal cells to enhance differentiation into osteoblasts and inhibit differentiation into adipocytes
  • 8. inhibits bone formation through a hypothalamic relay
  • 9. High expression of leptin was identified in hypertrophic chondrocytes in the vicinity of capillary blood vessels invading hypertrophic cartilage
  • 10. Leptin enhanced the proliferation, migration, tube formation, and matrix metalloproteinase-2 (MMP-2) activity of human endothelial cells
  • 11. leptin exerts its influence on endochondral ossification by regulating angiogenesis (creation of blood vessels) (The hypertrophic chondrocytes far from the blood vessels were negative for leptin)
Without lepton expression we are going to have both stunted growth and easily fracturable bones.

From Source Link HERE

Bone. 2005 Nov;37(5):607-21. Epub 2005 Jul 20.

Leptin regulates chondrocyte differentiation and matrix maturation during endochondral ossification.

Kishida Y, Hirao M, Tamai N, Nampei A, Fujimoto T, Nakase T, Shimizu N, Yoshikawa H, Myoui A.

Source

Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Japan.

Abstract

Leptin has been suggested to mediate a variety of actions, including bone development, via its ubiquitously expressed receptor (Ob-Rb). In this study, we investigated the role of leptin in endochondral ossification at the growth plate. The growth plates of wild-type and ob/ob mice were analyzed. Effects of leptin on chondrocyte gene expression, cell cycle, apoptosis and matrix mineralization were assessed using primary chondrocyte culture and the ATDC5 cell differentiation culture system. Immunohistochemistry and in situ hybridization showed that leptin was localized in prehypertrophic chondrocytes in normal mice and that Ob-Rb was localized in hypertrophic chondrocytes in normal and ob/ob mice. Growth plates of ob/ob mice were more fragile than those of wild-type mice in a mechanical test and were broken easily at the chondro-osseous junction. The growth plates of ob/ob mice showed disturbed columnar structure, decreased type X collagen expression, less organized collagen fibril arrangement, increased apoptosis and premature mineralization. Leptin administration in ob/ob mice led to an increase in femoral and humeral lengths and decrease in the proportional length of the calcified hypertrophic zone to the whole hypertrophic zone. In primary chondrocyte culture, the matrix mineralization in ob/ob chondrocytes was stronger than that of wild-type mice; this mineralization in both types of mice was abolished by the addition of exogenous leptin (10 ng/ml). During ATDC5 cell differentiation culture, exogenous leptin at a concentration of 1-10 ng/ml (equivalent to the normal serum concentration of leptin) altered type X collagen mRNA expression and suppressed apoptosis, cell growth and matrix calcification. In conclusion, we demonstrated that leptin modulates several events associated with terminal differentiation of chondrocytes. Our finding that the growth plates of ob/ob mice were fragile implies a disturbance in the differentiation/maturation process of growth plates due to depletion of leptin signaling in ob/ob mice. These findings suggest that peripheral leptin signaling plays an essential role in endochondral ossification at the growth plate.

Source Link HERE

J Histochem Cytochem. 2002 Feb;50(2):159-69.

Potential role of leptin in endochondral ossification.

Kume K, Satomura K, Nishisho S, Kitaoka E, Yamanouchi K, Tobiume S, Nagayama M.

Source

First Department of Oral and Maxillofacial Surgery, School of Dentistry, The University of Tokushima, Tokushima, Japan.

Abstract

Leptin, a 16-kD circulating hormone secreted mainly by white adipose tissue, is a product of the obese (ob) gene. Leptin acts on human marrow stromal cells to enhance differentiation into osteoblasts and inhibit differentiation into adipocytes. Leptin also inhibits bone formation through a hypothalamic relay. To obtain a better understanding of the potential role of leptin in bone formation, the localization of leptin in endochondral ossification was examined immunohistochemically. High expression of leptin was identified in hypertrophic chondrocytes in the vicinity of capillary blood vessels invading hypertrophic cartilage and in a number of osteoblasts of the primary spongiosa beneath the growth plate. The hypertrophic chondrocytes far from the blood vessels were negative for leptin. Moreover, we detected the production and secretion of leptin by a mouse osteoblast cell line (MC3T3-E1) and a mouse chondrocyte cell line (MCC-5) by RT-PCR, immunocytochemistry, and Western blotting. Leptin enhanced the proliferation, migration, tube formation, and matrix metalloproteinase-2 (MMP-2) activity of human endothelial cells (HUVECs) in vitro. These findings suggest the possibility that leptin exerts its influence on endochondral ossification by regulating angiogenesis.

PMID:   11799135         [PubMed – indexed for MEDLINE]

Leptin differentially regulates endochondral ossification in tibial and vertebral epiphyseal plates.

Longitudinal bone growth is governed by a complex network of endocrine signals including leptin. In mouse, leptin deficiency leads to distinct phenotypes in bones of the limb and spine, suggesting the appendicular and axial skeletons are subject to differential regulation by leptin. We established primary cultures for the chondrocytes from tibial and vertebral epiphyseal plates. Cellular proliferation and apoptosis were analyzed for the chondrocytes that had been treated with various concentrations of leptin. Crucial factors for chondrocyte proliferation and differentiation, such as BMP7 and Wnt3, were measured in the cells treated with leptin alone or in combination with pharmacological inhibitors of STAT and ERK signaling pathways. Primary culture of tibial epiphyseal plate chondrocytes has greater proliferating capability compared with that of vertebral epiphyseal plate chondrocytes. Leptin could promote the proliferation of tibial epiphyseal plate chondrocytes, while its effect on vertebral epiphyseal plate chondrocytes was inhibitory. Consistently, apoptosis is inhibited in tibial but promoted in vertebral epiphyseal plate chondrocytes by leptin. Importantly, leptin differentially modulates chondrogenic signaling pathways in tibial and vertebral epiphyseal chondrocytes through STAT and ERK pathways. Leptin differentially regulates chondrogenic proliferation and differentiation in appendicular and axial regions of the skeletons. The signaling pathways in these two regions are also distinct and subject to differential regulation by leptin through the STAT pathway in tibial epiphyseal plate chondrocytes but through the ERK pathway in vertebral epiphyseal plate chondrocytes. Therefore, the regulation of leptin is multi-faceted in the distinct anatomical regions of the skeleton. Knowledge gained from this system will provide insights into the pathophysiological causes for the diseases related to bone development and metabolism.”

Odd usually you think long torso shorter legs in higher body fat percentage people.

“Both the tibial and vertebral epiphyseal plates sustained the chondrogenic characteristics. We also confirmed that both tissues strongly expressed an important regulator of chondrogenesis, bone morphogenetic protein 7 (BMP7), a component in transforming growth factor β (TGFβ)
signaling pathway”

So what we’re actually looking for is a compound that decreases apoptosis in the vertebrae and the tibia.

Excessive Production of cGMP From Natriuretic Peptide Receptor Gene Mutation Leads To Tall Stature

Me: This is a continuation on the study of how cGMP and NPR2 effects skeletal growth and height. A type of mutation known as p.Val883Met in occurs in Npr2 which encodes the CNP receptor NPR2 (aka natriuretic peptide receptor 2). In a cell culture prepared in a certain way, the DNA with the specfic gene mutation generated intracellular cGMP (cyclic guanosine monophosphate) without the CNP ligand. With the ligand though, the cGMP production was higher in cells with the mutation. the cGMP was seen in the cartilage, which was also seen not only in the cell but also the family that was being analyzed. Blood sampling showed cGMP concentration to be high. The results concluded that the mutation of the NPR2 gene can possibly lead to increased cGMP production in the growth plates leading to larger than normal bone elongation. 

It seems that CNP (c-type natriuretic peptide) plays an influential role in chondrocyte development. When CNP as the ligand binds to the receptor NPR2, the NPR2 seems to act as Guanyl Cyclase which increases cGMP levels in chondrocytes. experimental mice that are bred with the specific mutated gene shows CNP overproduction and excess growth.

Quoted from the abstracts…

“”In human, overproduction of C-type natriuretic peptide (CNP) due to a chromosomal translocation was reported to cause skeletal dysplasia associated with tall stature. cGMP production downstream CNP/NPR2 system regulates the proliferation and differentiation of chondrocytes and determines skeletal growth.””

Since it would appear that only one single mutation of a specific gene can cause tall stature as exhibited within the family, that was the reason why the invention with the Guanyl Cyclase was probably patented. It is a form of gene therapy that has real legitimacy to work since gene therapy can specifically target individual genes using vectors. 

From source link HERE

PLoS One. 2012; 7(8): e42180.
Published online 2012 August 3. doi:  10.1371/journal.pone.0042180
PMCID: PMC3411678

An Overgrowth Disorder Associated with Excessive Production of cGMP Due to a Gain-of-Function Mutation of the Natriuretic Peptide Receptor 2 Gene

Kohji Miura,1 Noriyuki Namba,1 Makoto Fujiwara,1 Yasuhisa Ohata,1 Hidekazu Ishida,1 Taichi Kitaoka,1 Takuo Kubota,1 Haruhiko Hirai,1 Chikahisa Higuchi,2 Noriyuki Tsumaki,3 Hideki Yoshikawa,2 Norio Sakai,1 Toshimi Michigami,4 and Keiichi Ozono1,*

Abstract

We describe a three-generation family with tall stature, scoliosis and macrodactyly of the great toes and a heterozygous p.Val883Met mutation in Npr2, the gene that encodes the CNP receptor NPR2 (natriuretic peptide receptor 2). When expressed in HEK293A cells, the mutant Npr2 cDNA generated intracellular cGMP (cyclic guanosine monophosphate) in the absence of CNP ligand. In the presence of CNP, cGMP production was greater in cells that had been transfected with the mutant Npr2 cDNA compared to wild-type cDNA. Transgenic mice in which the mutant Npr2 was expressed in chondrocytes driven by the promoter and intronic enhancer of the Col11a2 gene exhibited an enhanced production of cGMP in cartilage, leading to a similar phenotype to that observed in the patients. In addition, blood cGMP concentrations were elevated in the patients. These results indicate that p.Val883Met is a constitutive active gain-of-function mutation and elevated levels of cGMP in growth plates lead to the elongation of long bones. Our findings reveal a critical role for NPR2 in skeletal growth in both humans and mice, and may provide a potential target for prevention and treatment of diseases caused by impaired production of cGMP.

Introduction

Several lines of evidence indicate that signaling triggered by CNP plays an important role in chondrocyte development [1], [2]. Upon CNP binding, its cognate receptor natriuretic peptide receptor 2 (NPR2) functions as a guanylyl cyclase to increase cyclic guanosine monophosphate (cGMP) levels in chondrocytes, female reproductive organs, and endothelial cells [3], [4]. Transgenic mice that overproduce CNP exhibit excessive growth, while defects of the CNP or Npr2 gene, leading to impairment of skeletal development [5]–[7]. The increase in cGMP level activates cGMP-dependent protein kinase II and seems to promote the accumulation of extracellular matrix in the growth plate of CNP-transgenic mice [8]. In human, overproduction of C-type natriuretic peptide (CNP) due to a chromosomal translocation was reported to cause skeletal dysplasia associated with tall stature [9]–[10]. In addition, acromesomelic dysplasia, type Maroteaux, characterized by dwarfism and short limbs, is caused by loss-of-function mutations in the Npr2 gene [11]. On the other hand, NPR3, which is thought to act as a clearance receptor, knock-out mice resemble CNP transgenic mice [12].

In this paper, we describe the first family with tall stature and macrodactyly of both great toes caused by a gain-of-function type mutation in the Npr2 gene. The mutant receptor, p.Val883Met, constitutively generates cGMP in vitro. Animal studies using the transgenic mice expressing the mutant NPR2 in chondrocytes demonstrated that skeletal overgrowth was associated with the overproduction of cGMP in cartilage. Our findings provide evidence that cGMP production downstream CNP/NPR2 system regulates the proliferation and differentiation of chondrocytes and determines skeletal growth.

Growth Plate Senescence Is Associated With Loss Of DNA Methylation.

Me: It would appear that the senescence of growth plates can not just be explained by one triggering step or mechanism like the idea that senescence of growth plates occur only from “growth plate senescence is caused by qualitative and quantitative depletion of stem-like cells in the resting zone” or that “senescence might occur because stem-like cells in the resting zone have a finite proliferative capacity, which is exhausted gradually”. I am sure that it is just one of the causes for growth plate eventual failure.

This new article that I have found seems to show the loss of DNA methylation is another main reason. I had found this article more than 2 months ago but at the time I did not understand what it was talking about so I had chosen not to read about it until now when I am more knowledgeable on the minute details on how the growth plates work. They have observed that the level of DNA methylation in resting zone chondrocytes decreased with age in vivo (within the lab animal). This drop seen in DNA methylation only occurs in the slow proliferation activity of chondrocytes in the resting zone of the animal, but nowhere else  as the rate of DNA methylation stayed the same from the resting zone, to the proliferative layer, to the hypertrophic layer. 

The conclusion reached is that the overall level of DNA methylation decreases during growth plate senescence. It agrees with the idea that (and I quote from the abstract 

hypothesis that the mechanism limiting replication of growth plate chondrocytes in vivo involves loss of DNA methylation and, thus, loss of DNA methylation might be a fundamental biological mechanism that limits longitudinal bone growth in mammals, thereby determining the overall adult size of the organism.

Then the obvious question would be what then causes the loss of DNA methylation? plus the other more practical question, if we can reverse or inhibit the decrease in rate of DNA methylation, can we keep the mechanism for the replication of growth plate chondrocytes in the resting zone constant or even increase in numbers and capacity?

From PubMed website. source link HERE.


J Endocrinol. 2005 Jul;186(1):241-9.

Growth plate senescence is associated with loss of DNA methylation.

Nilsson O, Mitchum RD Jr, Schrier L, Ferns SP, Barnes KM, Troendle JF, Baron J.

Source

Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. ola.nilsson@nih.gov

Abstract

The overall body size of vertebrates is primarily determined by longitudinal bone growth at the growth plate. With age, the growth plate undergoes programmed senescence, causing longitudinal bone growth to slow and eventually cease. Indirect evidence suggests that growth plate senescence occurs because stem-like cells in the growth plate resting zone have a finite proliferative capacity that is gradually exhausted. Similar limits on replication have been observed when many types of animal cells are placed in cell culture, an effect known as the Hayflick phenomenon. However, we found that the number of population doublings of rabbit resting zone chondrocytes in culture did not depend on the age of the animal from which the cells were harvested, suggesting that the mechanisms limiting replicative capacity of growth plate chondrocytes in vivo are distinct from those in vitro. We also observed that the level of DNA methylation in resting zone chondrocytes decreased with age in vivo. This loss of methylation appeared to occur specifically with the slow proliferation of resting zone chondrocytes in vivo and was not observed with the rapid proliferation of proliferative zone chondrocytes in vivo (i.e. the level of DNA methylation did not change from the resting zone to the hypertrophic zone), with proliferation of chondrocytes in vitro, or with growth of the liver in vivo. Thus, the overall level of DNA methylation decreases during growth plate senescence. This finding is consistent with the hypothesis that the mechanism limiting replication of growth plate chondrocytes in vivo involves loss of DNA methylation and, thus, loss of DNA methylation might be a fundamental biological mechanism that limits longitudinal bone growth in mammals, thereby determining the overall adult size of the organism.

PMID: 16002553         [PubMed – indexed for MEDLINE] 

READ THIS: Save 5 Minutes To Your Daily Schedule By Following This Tip!

This tip is for the regular readers who come to this website. I deeply care about who my regular readers are and I want to try to make this website as user-friendly and intuitive to navigate as possible. This is also why I want to give a tip to the readers of this blog that if you are the type who comes back often to the website hoping to find new article posts, you will probably be disappointed. I don’t spread out the writing of the posts randomly.

This is actually how my schedule really works. Most of the posts that I write comes out during the 12 noon-4 pm time range. I am currently in Seoul, South Korea so for the eastern or pacific time zones in the US, this corresponds to really  8 pm – 12 midnight in the Pacific Time Zone and around  11 pm – 3 am in the Eastern Time Zone. So if you want to be sure the next day’s or the current day’s new posts are out, if you are living in say Los Angeles, that would be at 12 midnight usually. For New York, that would be 3 am.

Of course I am not suggesting that a reader should stay up past 12 or 3 just to wait for the next article posting are out. Get your sleep. My articles and posts appear on a relatively consistent time. So save your time, which I guess is probably 3-5 minutes each day coming back to the website hoping new articles will be posted.

Be smart with your time and allocate this most precious well because it is one of the most important skills one must master as a professional responsible adult. Now have a great day guys!

The Changing Body And Increase In Height Of Japanese Women And Girls In The Last Century

Me: I wanted to focus this post on the changing form seen in the Japanese female over the last century. If we view the evolution of humans just from the perspective of height, we can guess at a reasonably accurate rate that humans have been getting healthier and living longer than at any other time in human history. From source link HERE

Changes in the physiques of Japanese women,       Much closer to their American sisters    —–     By T W Lim

Introduction. Every year since 1946, Japanese government, private sector companies and  foundations have carried out meticulous research of the Japanese physique and have discovered several startling statistics. Height, weight, susceptibility to cancer, female menstruation, and puberty  have been measured consistently. Japanese children now weigh as much as their adult counterparts (Weight loss surgery for children) after the WWII. Over a period of 10,000 years in Japanese history, from the study of archeological finds and records, Japanese had grown by only less than 10 cm . But within last 100 years, the record of last 10,000 years has been shattered. In the mid-1990s, Japanese girls experienced the biggest change at the primary school 6th grade level as they were taller than their postwar 1940s counterpart by nearly 16 cm.  As for female 7th graders, they are more than 12 kg heavier than their postwar 1940s counterpart.  Now, Japanese over 5 feet 7 inches or even 6 feet are very common.

One of the largest dietary change is probably consumption of milk. Not only has it made Japanese women grow taller, bigger and stronger but the consumption of milk has also advanced the age at which first menstruation happens in Japanese girls from 15 in 1950 to 12 years in 1975.  Only in the US has this record been somewhat matched and surpassed. Girls in the US are reaching puberty,  for example, as early as eight years old according to some reports and puberty is also accelerating though the national average is still about 12. This means that Japanese women have now reached the forefront of first world standards in female puberty. The implications need to be studied more.

In the same time period of 25 years after WWII, Japanese women have also grown more than 4 inches in height. Their weight increased by nearly 10 kg in the same period. Many scientists attribute this growth to the fact that Japanese consumption of dairy products now amounts to more than 50 kg per year, including cheese, milk, butter etc. Percentage of fat has also gone up with the intake of dairy products and that probably explains why Japanese women are now heavier.

American and Japanese researchers have identified over 50 hormones in milk that could possibly explain this phenomenon. The government and private sector has also played a key role in promoting milk,  making it readily available in offices, schools as well as vending machines in railway station. Hormones work in minute amounts and only a small tablespoon (or micrograms) of female hormones like estrogen or progesterone can produced a monumental effect on women’s growth.

Some researchers argue that there is a genetic potential for each race and that the Japanese are reaching their potential through improved diet but this is controversial and not readily proven. This is a controversial theory as it is not known if this so-called genetic potential may become a limiting factor on female growth in Japan. Would it be a genetic limitation such that Japanese women or indeed Asian women will never grow as large as Western women? Will the science of genetics or life sciences or biotechnology or genetically modified food in the future change this genetic potential? All questions remained unanswered.

Yet other researchers have gone beyond milk and look at soy proteins as a source of Japanese growth. These proteins consumed in larger amounts prevent menopausal problems but still promote proteins for growth. This finding is still controversial and it may be safer to say that Japanese are growing taller due to both intake of soy proteins as well as dairy products. There is evidence that even with the increase of soy proteins, there is a corresponding increase in animal proteins to more than 50 grams per day since 40 years ago. Studies have also shown that the soy proteins balance women’s hormones in Japan. These assertions of soy protein theory still need to be studied closely.

More Japanese women are also taking up aerobics, playing recreational sports like golf and strength sports like tennis, increasing their physique and musculature. More are training harder and earlier and Japanese educational sports programs are known to be rigorous and strict. In most other East Asian nations, women cannot afford the leisurely time to play such sports and have less time to exercise than Japanese women, especially in Asian countries which are barely surviving on their food supplies like India, Indonesia which are seeing a large part of their populations starving.

Some in the West wondered how Japanese women who are constrained to small spaces in Japan can grow big and tall suddenly. Wealth has also overcome Japanese space constraints as more and more Japanese women go to ultra-modern gyms and sports clubs to train their muscles. Taut, veined athletic muscles are no longer frowned upon in the Japanese fashion world thanks to fitness trends in the US, a source of Japanese emulation.There are other more minor theories like Japanese moves from sitting on the floor to sitting on chairs, have improved their postures and musculatures accounting for the height and weight increase. Removal of infectious diseases, wealth and better quality of living as well as Japanese infatuation with disinfectant may also be promoting Japanese growth, size and longevity. Other less scientific assertions include more sleep for Japanese improving their metabolism rates. Others have attributed the growth to the many calorie-, vitamins- and minerals-enhanced food and drinks available from vending machines which youngsters drink on their way to night prep schools in place of dinner due to time constraints.

Besides diet, some Japanese researchers and scientists say increase may also be due to Japanese industrial economy producing large amounts of chemical byproducts that are artificially increasing the sizes of women. Dioxins and polystyrenes have been identified by some as chemical substances that may enlarge and extend Japanese female body sizes. They are hormone effectors that have been found in Japanese soil, river and seas. These substances in Japan have been blamed for low sperm counts and making men develop feminine characteristics. Synthetic estrogens have already caused changes in fish and frogs in the rivers.

Finally, some of these changes in Japanese female physique may also be due to Japan’s state intervention. Some schools for example have their kids or encourage their kids to taking vitamins pills in specially prescribed boxes during lunchtime or have arranged volumes of milk or dairy products to promote growth. Others have prescribed rest time to manage youngsters’ metabolic rates. Schools strictly monitor young children’s diet (Weight loss surgery for children) with regular school inspections of what mothers put in their bento or lunch boxes. They regularly prescribe diets with calorie intakes provided for the parents. Parents also have been educated more through the media and during parent-teacher meetings diets are often discussed to standardize growth promoter diet for Japanese children.

Psychological Factors.

Psychologically, Japanese who are afraid that they will stick out because of their height or find it difficult to marry no longer feel like that. Tall women are increasingly envied. In fact, they are so admired that at one time, many Japanese women wear high heel or platform shoes to make themselves look taller, even to the extent of towering over their boyfriends who now have to live with such fashion.

Another psychological factor studied by some social science and psychology researchers in Japan is the growing assertiveness of Japanese women. They are becoming more aggressive and independent compared to their traditional demure image. It is not known if this is due to biological factors. But women are seen as stronger in careers and personalities. Could chemical compounds be responsible for this change?