Puberty

Question 1

Define puberty.

Answer 1

• Complex developmental event
• Continuum of changes leading to somatic and sexual maturation
• Profound physiological, psychological and physical changes
• The main goal from a reproductive perspective is the attainment of sexual maturity or fertility. For males, that is the production of sperm, and, for females, it is the development of meiotically competent oocytes. Need to produce mature gametes = spermatozoa from the testes, oocytes from ovaries.
  Females are born with their full oocyte complement. When puberty kicks in, it is the development of these oocytes to the point where they are competent for fertilisation.
• Breast development in females, and increased testicular volume in males
  From a clinical perspective, when looking at puberty, it is defined as breast budding in females and increased testicular volume in males (>4ml).

Question 2

What are the two endocrine events of puberty?

Answer 2

1) The first endocrine event is known as adrenarche, resulting in the production of adrenal androgens, and is responsible for the growth of pubic hair, axillary hair and growth in height
2) The second endocrine event is gonadarche, which is essentially the switching of the HPG axis. It results in the secretion of gonadotrophins = FSH and LH. LH is important in steroid synthesis for the development of secondary sex characteristics. FSH is required for growth of testis (male)/steroid synthesis/ folliculogenesis (female)
- These two endocrine events culminate in puberty; It is important to note that these two events are independently regulated. Adrenarche is not required for gonadarche to occur and vice versa. They occur independently of one another.

Question 3

What is adrenarche?

Answer 3

• First endocrine process of puberty
• Occurs ~6-8 years. Surge from around ages 6-8, Peak at mid 20s and then a gradual decline.
• Characterised by (re-)instigation of adrenal androgen secretion =
  1) Dehydro-epiandrosterone (DHEA)
  2) Dehydro-epiandrosterone sulphate (DHEA-S)
• There is a remodelling of the adrenal cortex that occurs in order for it to be able to secrete DHEA and DHEA-S (androgens).
• DHEA and DHEA-S secretion is from the zone reticularis as the innermost layer of the adrenal cortex. Result of inherent maturation of cellular compartments of adrenal cortex.
• Studies have shown there is no change in cortisol/other adrenal hormones during adrenarche (just DHEA and DHEA-S) - not a global activation of HPA axis

Question 4

How do the adrenal glands remodel during development?

Answer 4

• Remodelling begins at the foetal stage. In the foetus, the adrenal gland has two zones = foetal zone and the definitive zone. The foetal zone is responsible for DHEA and DHEA-S production in the foetus.
• Once the foetus develops and is born (neonate stage of development), there is an involution (shrinkage) of the foetal zone. This is accompanied by a drop in DHEA and DHEA-S production.
• The definitive zone starts to expand and differentiate into the layers of the adrenal cortex that adults have = zona glomerulosa and zona fasciculata. At this infant stage, the differentiation of these two outer layers of the adrenal cortex occurs and, at this point, DHEA and DHEA-S production is switched off.
• At about age 3, focal islands of zone reticularis (distinct patches) develop.
• From ages 4 and 5, these patches start to expand and come together to form a continuous and functional zona reticularis layer.
• Once this layer is fully functional and developed at age 6, DHEA and DHEA-S production is switched on again but now the remodelling is complete. Age 6 years = functional ZR developed and androgen production resumes. So before it was the foetal zone in the foetal adrenal gland, now remodelling is complete and it is the ZR that is producing the adrenal androgens. All through to puberty (12-13 years), the ZR layer continues to expand and continue to see a rise in DHEA and DHEA-S secretion.
• Once the remodelling is complete, it kicks off adrenarche; DHEA and DHEA-S production after remodelling is the start of adrenarche (~age 6-8).

Question 5

Compare the histology of the adrenal gland in an infant and in an individual undergoing puberty.

Answer 5

• The medulla is outside of the cortex. It is followed by the three layers of the cortex. Firstly, the ZR.
• Larger and more robust ZR in the individual undergoing puberty compared to an infant.
• Followed by the zona fasciculata and then the zona glomerulosa.

Question 6

How is DHEA/S made?

Answer 6

• Made via a series of steroidogenic conversions. Cholesterol is the original precursor; there are a series of conversions that occur. Eventually culminate in the production of DHEA and DHEA-S. Done with the aid of steroidogenic enzymes, particularly CYP11A which is responsible for the conversion of cholesterol to pregnenolone (the second precursor). CYP17 is responsible for DHEA production, while SULT2A1 is responsible for making DHEA-S from DHEA.
• Looking at histology sections (immunohistochemistry), an individual undergoing puberty has increased expression of CYP11A for that initial conversion of cholesterol to pregnenolone in the ZR compared to that of an infant (pre-pubertal). The same applies to CYP17.
• There is a pathway responsible for the synthesis of glucocorticoids and mineralocorticoids, e.g. cortisol, cortisone. Usually, after the initial conversion to pregnenolone, there is a diversion through the sideways pathway (modulated by 3- beta-HSD). It converts pregnenolone and hydroxypregnenolone into the corticosteroids. In pubertal individuals, that is virtually no expression (a significantly reduced expression) of 3-beta-HSD compared to that of an infant. This is because, during adrenarche, the sideways pathway is shut down so that the cholesterol and pregnenolone are committed (concentrated) towards making DHEA and DHEA-S and none of it is lost in the sideways pathway.
• There is increased expression of SULT2A1 in an individual undergoing puberty compared to that of a pre-pubertal infant.
• Important summary = During adrenarche, there is increased expression of SULT2A1in the ZR of an individual undergoing puberty, because that is responsible for DHEA-S production, and there is reduced (if not switched off) expression of 3-beta-HSD, which prevents the loss of pregnenolone and cholesterol to the sideways 3-beta-HSD pathway.

Question 7

What is the function of DHEA/S?

Answer 7

• Synthesised by the adrenal gland (in the ZR of the adrenal cortex)
• Transported by the bloodstream and converted into DHT in the peripheral tissue. Metabolism within peripheral tissue. Particularly the production of DHT which drives the maturation of hair follicles required for the growth of pubic and auxiliary hair.
• DHT is responsible for the growth of pubic hair, axillary hair, the change in skin glands and prostate secretions.

Question 8

What instigates adrenarche?

Answer 8

1) ACTH?
- The first piece of evidence that made scientists want to look at ACTH was dexamethasone (synthetic corticosteroid).
- Dexamethasone suppresses adrenal androgen production.
- Children with ACTH receptor mutations fail to undergo adrenarche.
- But, no change in ACTH/cortisol during adrenarche.
- Further studies revealed that children with ACTH receptor mutations fail to undergo adrenarche. However, since the levels don’t change during adrenarche, even if there is any involvement at all, it can’t be the trigger/driving factor.
- Divergent mechanisms for cortisol and androgen production at adrenarche?

2) POMC?
- The second candidate that was proposed is pro-opiomelanocortin (POMC) = 241AA sequence that undergoes cleavage into multiple peptides.
- Proximal 18 AA region that positively regulated adrenal androgen production.
- In vitro studies did not substantiate this
- The initial findings supporting the positively regulated adrenal androgen production was a result of many in vivo studies, however the in vitro and cell culture studies did not substantiate this so POMC was dropped as a candidate.

3) POMC-related peptides?
- POMC-related peptides were looked at due to mere correlations, but no concrete evidence existed/was found
- b-lipotrophin and b-endorphin plasma levels correlate with increased DHEA/S at adrenarche

4) Other factors ruled out include prolactin, IGF-1 and insulin
- No conclusive mechanism for control ofadrenarche.

Question 9

What is gonadarche?

Answer 9

• Characterised by (Re-)Activation of HPG axis. Gonadarche is modulated via the archetypal HPG axis paradigm.
• Usually occurs several years after adrenarche (typically ~11 yrs of age).
• Driven by hypothalamic GnRH & pituitary gonadotrophins.
• The hypothalamus releases GnRH which stimulates the anterior pituitary to release gonadotrophins (LH/FSH). This results in steroidogenesis in the gonads.
• Puberty depends on reactivation of GnRH release!

Question 10

What is the role of the HPG axis involved in puberty?

Answer 10

• GnRH is synthesised & secreted → Synthesis and secretion of pituitary gonadotrophins (LH & FSH) →
  Gonadal steroid production →
  Negatively/positively feedback onto hypothalamus-pituitary to regulate GnRH and LH/FSH production
• The ovaries produce egg cells. The steroids produced result in the changes involved in puberty. Oestrogen = broadening of hips, menstruation begins, development of breasts, pubic and axillary hair. Testosterone = voice “breaks”, growth of muscle tissue, enlargement of genitalia, facial, pubic and axillary hair.

Question 11

How is GnRH released during development to puberty?

Answer 11

• During the 16th gestational week of development, the HPG axis in the foetus is activated for the first time. Pulsatile GnRH secretion in foetus and 1-2 years postnatal increased. This is what is responsible for the production of the HPG axis hormones during that period of development. It continues until just before birth where it is switched off (believed to do this to make way for the feedback from placental hormones). Then, after birth, HPG axis switched on for one to two years postnatally before being switched off again.
• In the week 12 pregnancy scan, the sex of the baby cannot be definitively determined but can be in the 20 week scan. This is because of the 16th week activation of the axis which is required for completion of sexual differentiation.
• It is believed that after it is switched off following the post natal period, the neurons are restrained (called a restraining of the GnRH neurons) = 10 years or more
  . The reason it is switched on again after birth is not known but it is believed to be a priming of the HPG axis. Prior to birth, it is used for the foetal physiology and modulation of the axis. After birth, it is an initial priming to get the HPG axis used to its function.
• At puberty, there is a gradual rise in pulsatile release of GnRH. Tends to occur around one year before some secondary sexual characteristics, e.g. breast budding, start to be seen.
• Reactivation usually begins with a nocturnal rise in GnRH levels (start to pick up at night). During puberty, there are more consistent levels across the day. As an adult, the full menstrual cycle is in play and there are cyclical differences. In males, there is a more consistent pattern. This is measured using LH levels.

Question 12

Describe consonance through the Tanner stages of puberty.

Answer 12

• The movement through the stages = consonance. This progression is the same in every individual; it is the time spent in each stage and the time it takes to move from one stage to another that differs
• A paediatric endocrinologist, Tanner, created the Tanner stages of puberty. It is a very useful to in detecting when puberty is out of consonance. This is how pubertal disorders, such as hypogonadotrophic hypogonadism or Kallmann’s syndrome, are picked up.
• The stages progress from 1 to 5 looking at breast development in girls, pubic hair development in both sexes and genital development in boys.
• Physiological changes occur at different stages. Menarche (onset of menstruation) doesn’t begin until Tanner stage 4, as opposed to males where sperm production begins at Tanner stage 2.

Question 13

What are the current theories surrounding the onset of puberty?

Answer 13

• A number of theories have been put forward regarding onset of puberty:
  1) Inherent maturation of CNS
  2) Body fat/nutrition – Leptin and Ghrelin?!!
  3) Hypothalamic hormones – Kisspeptin (especially), other factors?
  4) Latest theories - Epigenetics? Epigenetics – changes in the expression/phenotype of a gene without any changes in the sequence, e.g. methylation. These changes may be triggered by environmental factors and other exposures. They can be passed down to offspring. A lot of the theories on epigenetics point to the narrative that parents’ lifestyle and diet had epigenetics effects on the genes that they passed down to future generations – where the DNA sequence wasn’t changed, the way it was expressed was changed as a result of certain exposures.
• In terms of the latest theories, epigenetics is a big one. This is something that is being actively researched currently.
• There have been some theories and hypotheses with regards to body fat and nutrition (along with actual evidence).

Question 14

What are the potential adverse risks from early puberty?

Answer 14

• cardiovascular disease
• metabolic disease
• obesity
• diabetes
• disordered behaviour
• decreased adult height (linked with early puberty because bone fusion occurs much earlier than normal). Bone fusion marks the end of a growth in height, so the individual does not grow as tall as they could have.
  Sexual and psychological maturation may be out of sync (individual is undergoing sexual maturation way before psychological maturation) in early puberty; can lead to potential mental health problems.
• decreased life expectancy. All of these come with a decreased life expectancy, which is why puberty is an important area to focus on from an epidemiological perspective.

Question 15

What controls the onset of puberty?

Answer 15

• Dialogue between our individual genetics and environmental factors
• All impinge at different points of the HPG axis

Question 16

When is the onset of puberty?

Answer 16

• Secular trend to earlier age at menarche in girls from W. Europe & USA
• Average age of menarche onset (UK) is 12.5yrs
• Data from different countries in the Western world shows the age of onset of puberty over the last 100-150 years. Stark uniform decrease in the age of onset (decreased from 17 years old to now 12 and lower). A common denominator here is that we are now a lot better fed (also more lethargic as a result). This suggests a link between energy homeostasis and reproduction.
• Changes in age at menarche, 1840 to 1978, illustrating the advance in the age at menarche in Western Europe and the United States since 1840 and the slowing of this trend since about 1965. (Modified from Tanner M, Eveleth PB. Variability between populations in growth and development at puberty. In Berenberg SR, ed. Puberty, Biologic and Psychosocial Components. Leiden: HE Stenfert Kroese, 1975:256-273.)
• Link between energy homeostasis & reproduction

Question 17

What is the impact of nutrition and body fat on puberty?

Answer 17

• Extremes of energy excess (body fat mass) impact the timing of puberty in both sexes - particularly females
• Under- and over-nutrition in foetal and/or neonates alters the timing of puberty in rodents and humans
• Morbid obesity (females) can cause precocious puberty
• A series of studies carried out in the 70’s/80’s by Frisch et al. lead her to postulate this hypothesis. Frisch et al.: “critical fat mass” hypothesis
  Threshold % fat/body weight is required to attain (17%) and maintain female reproductive ability (22%)
• Cross talk between metabolic status and puberty onset/maintenance of reproduction
• A lot of studies that we have are in females; not as many studies in males (deficit of evidence relating to males).
• Morbid obesity in females can cause early puberty.
• From an evolutionary perspective, this hypothesis (threshold body fat) makes sense as pregnancy and lactation are energy-intensive processes. Energy reserves are required; without them, you should not be reproducing. Also, cholesterol (body fat) is the precursor of all steroid hormones.

Question 18

What is the effect of nutritional gating on puberty?

Answer 18

• Based on the evidence available, we believe that secretions from the gut and adipose tissue act on the hypothalamus and, in turn, modulate gonadal function via the HPG axis.
• There is evidence available to support this, beginning with leptin.

Question 19

What experimental evidence confirms the role of leptin?

Answer 19

1) In the 50s, a mouse was isolated from a colony for its erratic behaviour. After a series of tests, it was found to be hyperphagic (because it ate a lot) but, most importantly, infertile and leptin deficient (had a spontaneous mutation in the gene coding for leptin) = natural version of a knockout mouse for leptin. (Ob/ob mouse)
- Hyperphagic
- Hyperglycaemic
- Insulin resistant
- Infertile
- Leptin deficient
2) On the human side of things, there was a group in Cambridge that was carrying out a research study that they named “Genomics of obesity”. Prof. Sadaf Farooqi’s group at Cambridge Uni. identified one of the first leptin mutations in human.
They were trying to look at the genetic causes and effects of obesity. In the process of carrying out this study, they came across this south American boy who was weighing 42 kg at 3yo (the average 3yo should be weighing anywhere between 12 and 20kg). The first human form of a leptin gene was cloned in 1994; expressed in white adipose tissue.
- 1994: leptin gene cloned
- The morbidly obese boy was leptin deficient (always hungry); found that he had a mutation in the gene coding for leptin when carrying out their research. Circulating levels directly proportional to amount of body fat; commonly believed (based on evidence) that in more common forms of obesity, there is lots of leptin but the leptin receptor is not responding properly/at all to the leptin signal. There is loads of leptin, but the receptor is not picking up that ligand so the brain is ultimately not getting the satiety signal. In the case of this boy, because he is leptin deficient, his brain is not getting the satiety signal (always feels hungry, brain thinks it has no energy reserves). Leptin deficiency = no satiety signal = keeps eating = morbidly obese. Common denominator = lack of satiety signal.
- Less than 10 families in the UK with this condition.
- Mice and humans with leptin deficiency due to mutation in gene present with hypogonadotrophic hypogonadism.

Question 20

What is leptin and how does it influence the reproductive system?

Answer 20

• Expressed in adipocytes- WAT
• Sensor of energy sufficiency
• Satiety factor - tells brain you’re full
• Stimulates energy expenditure
• Circulating levels directly proportional to amount of body fat
• Influence on reproductive system:
  1) ob/ob mice & humans - hypogonadotrophic hypogonadism
  2) Delayed/absent puberty
  3) Can be reversed with leptin injection
  4) Some leptin-deficient patients have normal menses/LH/oestradiol levels- unknown why (shows there are no clear-cut clinical cases).

Question 21

Is leptin the trigger to puberty?

Answer 21

• Sexual dimorphism:
  1) Females- rise ~ 2 years prior to puberty (increased GnRH pulsatility)
  2) Males- no rise
• There is a difference in males and females regarding the expression of leptin. In females, there is a rise in leptin levels about two years prior to puberty (accompanied by increased GnRH pulsatility).
• Expression of leptin is different in males and females.
• Obesity increases leptin and earlier puberty occurs (particularly in females)
• KO leptin in rodents and humans (mutations in leptin gene)- delayed/absent puberty
• BUT, leptin administration can not stimulate early puberty
• No leptin receptors on GnRH neurons
• Threshold of leptin required to be reached for puberty but not a driver of puberty itself.
• Leptin has a permissive role on puberty onset; suggests a threshold needs to be met but it is not the ‘driver’ of puberty.

Question 22

What is ghrelin and how does it influence the reproductive system?

Answer 22

• Gut peptides: Ghrelin (exact opposite of leptin)
• Ghrelin senses the fasted state, to stimulate feeding and fat deposition.
• A bolus of ghrelin stimulates the GH/IGF axis via GHSR. GHSR = Ghrelin receptor
• GH/IGF axis responsible for bone growth
• GHSR = growth hormone secretagogue receptor.
• In ‘starvation’ (high ghrelin) decreased activity of the HPG axis.
• Starved state = high levels of ghrelin = decreased activity of HPG axis. From an evolutionary perspective, should not be reproducing with low energy levels.
• Ghrelin decreases as puberty proceeds.
• Ghrelin can decrease hypothalamic kiss1 expression in rat (Forbes et al., 2009).
• Ghrelin can decrease expression of kisspeptin in rats. It is these pieces of evidence that started to make scientists look closer at ghrelin to see if it has a significant role in puberty.
• Subset of kiss1 neurons in selective hypothalamic nuclei that express GHSR (ghrelin receptor) and respond to Ghrelin.
• Oestradiol can also increase GHSR expression and response to Ghrelin in kiss1 neurons (Frazao et al., 2014). Can modulate the ghrelin receptor activity.
• Low levels of leptin and high levels ghrelin → decreased LH expression.

Question 23

Where are kisspeptin receptors expressed?

Answer 23

• Co-expression of GnRH mRNA with Kiss1R mRNA in rats from the medial preoptic area.
• ¾ of the GnRH neurons co-express Kiss1R mRNA .
• In situ hybridisation (used to show mRNA expression). Can see kisspeptin mRNA expressed in red clusters. Shows kisspeptin receptors are directly expressed on GnRH neurons, however it is important to know that this is at the mRNA level, so it doesn’t account for the translation into protein. Doesn’t tell you the availability of the receptor at the surface and the turnover rate at which it will be translated.
• Kisspeptin expression is found in the AVPV and arcuate nucleus

Question 24

What is the effect of continuous kisspeptin infusion on LH release in juvenile rhesus monkeys?

Answer 24

• This study was carried out by Tony Plant’s group in the US using rhesus monkeys..
• Old name = metastin
• They initially introduced a bolus of GnRH and that was characterised by a pulse in LH (spike). Then, they also introduced a bolus of kisspeptin as well and another spike was seen. This was followed up with a continuous administration of kisspeptin; resulted in a drop in LH levels (practically switched off)

Question 25

What is the effect of pulsatile kisspeptin administration on LH secretion in juvenile rhesus monkeys?

Answer 25

• Started by introducing pulses of GnRH which resulted in pulses of LH. Followed up with pulses of kisspeptin (introduced at intermittent intervals, similar to what would occur in a pulsatile situation) and the response as well was pulsatile LH.
• Shows that pulsatility is also crucial for activity of kisspeptin.

Question 26

What roles of kisspeptin have been associated with puberty?

Answer 26

• Original observations in GPR54 null mice / humans (mutations)
  1) Abnormal development of GnRH neurones → hypogonadism
  2) Failure to enter puberty
  3) KO mice for GPR54 or kisspeptin → hypothalamic hypogonadism
  4) Mutations in humans- hypothalamic hypogonadism
  5) Activating mutations of GPR54 → precocious puberty
  6) Phenotype (mice) –
  Male: small testes and epididymis, delayed spermatogenesis infertility;
  Female: small oviducts, folliculogenesis-no progression to ovulation, no oestrous cycles, infertility
• A lot of this evidence that we have is a result of studies that were carried out on KO mice for kisspeptin receptor and humans with mutations of the receptor.
• They present with hypogonadism (pubertal disorders)
• KO mice for kisspeptin receptor of kisspeptin itself result in hypogonadotrophic hypogonadism. Same for humans with either (mutations).
• Activating mutation of the receptor means it’s activated without the ligand present.

Question 27

What are the effects of starvation on the expression of Kisspeptin in the hypothalamus of pubertal and adult mice?

Answer 27

• Lots of research has been carried out to answer this
• Compared pubertal expression of kisspeptin in the starved state with adult. In the fasted state, there is decreased expression of kisspeptin when compared to the adult. This marked reduction still points us in the direction of nutritional gating.
• Only in pubertal mice is the level of expression markedly reduced; not in the adult mice

Question 28

How is the Kisspeptin-GnRH system integrated with metabolic cues (leptin)?

Answer 28

• Reduced leptin in starvation results in decreased GnRH secretion
• Leptin directly excites Kiss1 neurones in ARC
  Leptin deficiency is associated with ↓Kiss 1 mRNA expression in ARC
  But only 10-40% of Kiss1 neurones express LepR
• Indirect and direct mechanisms of Leptin action in hypothalamus on HPG axis
• There have been new proposed mechanisms of interplay between energy homeostasis and HPG axis. Modified version of HPG axis with additions. Includes gut hormones and adipose hormones.
• The factors in bold print that are interacting with the HPG axis have more concrete data to support their role in modulating the HPG axis, e.g. leptin, ghrelin. The factors in smaller print just have data that show correlations (resistin, chemerin).

Question 29

What switches on puberty?

Answer 29

• An integration of central and peripheral inputs determined both by genetics and environment/nutrition