2014 Group Project 4

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2014 Student Projects
2014 Student Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7 | Group 8
The Group assessment for 2014 will be an online project on Fetal Development of a specific System.

This page is an undergraduate science embryology student and may contain inaccuracies in either description or acknowledgements.

Genital

System Development

[NB: having formatting issues trying to put the following information into a table, an attempt at the table is included below]

  • the initial stages of genital development are indifferent, both males and females are anatomically identical until morphological changes occur beginning at week 7
  • males and female changes are determined by the chromosomal sexual differentiation which occurs at fertilisation
  • initial stages of gonadal development begins during the fifth week of development. First change is a thickened area of mesothelium on the medial side of the mesonephros [primitive kidney]
  • this proliferation + underlying mesenchyme produces a bulge known as the GONADAL RIDGE
  • finger like epithelial chords [gonadal chords] grow into underlying mesenchyme
  • This who structure is referred to as an indifferent gonad
  • indifferent gonad consists of an external cortex and internal medulla
  • in embryos with an XX sex chromosome, the cortex of the indifferent gonad progresses to developing into an ovary
  • Conversely, embryos with an XY chromosome, the medulla of the indifferent gonad progresses into a testes
  • CHROMOSOMAL SEX DETERMINATION - the indifferent gonads differ after the seventh week by the presence the SRY gene which produces the TESTES DTEREMINING FACTOR on the short arm of the Y Chromosome. Under this influence, the gonadal chords differentiate to somniferous cords expression of Sox9 and Fgf9
  • once the gonad that develops [ovary or testes] then determines the type of sexual and genital differentiation which occurs
  • fetal testes produce testosterone, dihydrotestosterone and anti mullerian hormone
  • TESTES DEVELOPMENT -- insert picture here

- seminferous chords --> rete testes - development of dense tunica albiguinea- enlarging testes separate from the degenerating mesonephros to form the somniferous tubules, tubuli, recti and rate testis - seminferous tubules --> interstitial cells [leydig cells] which produce androgens - insert diagram showing the production of androgens (testosterone, dihydrotestosterone and antimullerian hormones (sertoli cells) )

  • DEVELOPMENT OF OVARIES

- ovary not identifiable until 10th week - gonadal chords aren't prominent in developing ovary but extend to medulla and form the rate ovary OOGENESIS DIAGRAM

  • DEVELOPMENT OF GENITAL DUCTS
  • fifth and sixth weeks - mesonephric ducts (wolfing duct) and paramesonephric ducts (mullein ducts)
Weeks M A L E F E M A L E
FERTILIZATION both male and female are same at this point- only difference is presence of XY or XX chromosome
WEEKS 1-7 GENITAL DEVELOPMENT IS UNDIFFERENTIATED. gonads derived from:
* mesothelium lining posterior abdominal wall
* underlying mesenchyme
* primordial germ cells.
WEEK 5 development of indifferent gonads
  • thickened area of mesothelium develops on medial side of mesonephros [primitive kidney]
  • this + proliferation of underlying mesenchyme = gonadal ridge
  • fingerlike epithelial chords = gonadal chords
  • gonad = cortex + medulla
WEEK 8 seminiferous tubules begin to release androgens
WEEK 10 rudimental rete ovarii forms from indifferent gonads
WEEK 12
  • testosterone determining factor induces seminiferous chords from indifferent gonads --> branch to rete testis
  • tunica albiguinea develops
WEEK 16 primordial follicles begin to develop

Genital system development is an extremely interesting area of embryology as it is not until the later stages of embryogenesis (around week 4-6) that sexual differentiation occurs in the fetus, and the sexual organs actually look very similar up until this point, and the formation of the correct sex organs depend really on whether the genital ridge releases Testosterone or oestrogen

<pubmed>24240231</pubmed> <pubmed>24928207</pubmed> <pubmed>24741072</pubmed>

File:Image.jpg
The stages in sexual differentiation of the female and male reproductive system


Related video

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>


Current Research, Models and Findings

Current Research and Findings

Male

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.[1]


Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent. (2013)[2]

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:

  • Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
  • Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
  • Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
  • Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males. Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium. The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells. Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis. Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.


FEMALE

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Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)[3]

<pubmed>21584884</pubmed>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.[4]

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.


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In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)[5]

<pubmed>21584884</pubmed>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.


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Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)[6]

<pubmed>21223560</pubmed>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development [7]

Some critical genes involved in the testicular and ovarian pathways include:

  • SRY gene (sex-determining region of the Y-chromosome) [8]
  • Rspol gene (R-spondin homolog)[9]
  • Wnt4 – wongless-related MMTV integration site 4)[10]
  • Beta-catenin[9]

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.[9]

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.

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Other current research and findings:

  • A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. [11]Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). [12]


Current Models

Current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. [1]

Also, when observing the morphology and cell biology of the developing testis, it is important to note that much of the research conducted on the subject involved the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy.[13]


Current model: of the cellular and molecular mechanisms of the development of mammalian external genitalia. [1]

DEVELOPMENT OF EXTERNAL GENITALIA IN THE HUMAN

Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE

  • The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings.
  • These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal.
  • The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane.
  • These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.
  • These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly and the rectum and anus posteriorly. [1]
  • This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus. [1]

DRAW A PICTURE

  • When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
  • The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.

Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION

  • Initially, the female and male fetuses’ external genitalia are identical and includes the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally). [1]
  1. 1.0 1.1 1.2 1.3 1.4 1.5 <pubmed>14641326</pubmed>
  2. <pubmed>24030937</pubmed>
  3. <pubmed>21584884</pubmed>
  4. <pubmed>18431748</pubmed>
  5. <pubmed>16585961</pubmed>
  6. <pubmed>21223560</pubmed>
  7. <pubmed>19027189</pubmed>
  8. <pubmed>2247149</pubmed>
  9. 9.0 9.1 9.2 <pubmed>18250098</pubmed>
  10. <pubmed>18250097</pubmed>
  11. <pubmed>19379463</pubmed>
  12. <pubmed>8882299</pubmed>
  13. <pubmed>17237341</pubmed>