The Biology of Sexual Differentiation

Here, we are discussing physical and physiological differences between males and females.  In the past, there were some incorrect assumptions made about the processes involved in the determination of sex... the processes which make one person male, and another person female.  Notably, male scientists, perhaps in a male dominated scientific community of the past, correctly concluded that the determination of maleness was an "early, active process."  Having identified what was thought to be the gene responsible for formation of the testes in the 1950's (this was later shown to be the wrong gene), with no similar gene having been found for formation of the female ovary, it was incorrectly assumed that to become a female was a late, "default" process.  That is, if nothing happened to cause a fetus to become male, it became female by "default."

PART A:  Chromosomal Sex (Genetic Sex, Genotypic Sex)

Normal human diploid (2n) cells contain 22 autosomal pairs of chromosomes and 2 sex chromosomes (X X or X Y).  The human female karyotype is 46,XX and the human male karyotype is 46,XY, denoting a total of 44 autosomal chromosomes plus the 2 sex chromosomes.  The dyes most commonly used to look at karyotype are Giemsa stain and the fluorescent dye quinacrine.  The autosomes (non-sex chromosomes) are numbered according to decreasing length... ie. the longest autosomal chromosome is Chromosome I.

Chromosomal (genotypic, genetic) sex is determined at conception, with the contribution of an X or Y chromosome from the father.  The mother can contribute only X, no matter what the sex of the offspring.  For this reason, we often say that "the father determines the sex of the offspring," of course, this is a random event.

Sometimes, genotypic sex does not fit the typical pattern of XX (female) or XY (male).  Occasionally, because of a failure in meiosis during development of gametes, sperm or oocytes may be diploid... XX from the female, and XY from the male.  If, for example, an XY sperm fertilized an X oocyte, an embryo might become XXY (Klinefelter's syndrome).

Click here to see some examples of disorders of gentotypic sex

Then, come back and start reading here!

Gene Dosage Compensation:  A process called "gene dosage compensation" is thought to occur in anyone with 2 or more X chromosomes.  Gene dosage compensation means that all of the X chromosomes but one are randomly inactivated during the early developmental stages of the embryo.  Despite this, it appears that 2 X chromosomes are required for successful formation of ovaries.  Two copies of the X chromosome appear to be necessary for primordial germ cell migration in the female embryo... after which complete inactivation of the second X chromosome may be possible.  The result of inactivation of an X chromosome is formation of a sex chromatin body (Barr body) in the interphase cells of persons having 2 or more X chromosomes.  A sex chromatin body (or Barr body) is a condensed mass of chromatin containing a discarded X chromosome.  The sex chromatin body is usually observed near the periphery of the nucleus of an interphase cell.

A sex chromatin body (Barr body) is evident in 20-30% of normal 46,XX females and is absent in normal, 46,XY males. In patients with more than 2 X chromosomes, the number of sex chromatin bodies (Barr bodies) in any diploid (2n) nucleus is generally one less than the total number of X chromosomes they carry.  By examining sex chromatin (Barr bodies) and fluorescent staining of the Y chromosome, one can determine the sex chromosome complement of any individual.  That bolded statement probably sounds silly, but recognize that some individuals are born with ambiguous genitalia (that is, it is difficult to immediately determine, from their external genitalia, whether they are male or female) and it is sometimes necessary to make a "best genetic determination" of sex, or to diagnose the presence of a genetic disease involving the sex chromosomes.

sex ratio = ratio of males:females

Given that we have a heterogametic sex with 2 sex chromosomes, X and Y, what should be the normal genetic sex ratio at conception (theoretically it is 1:1)? ie. Mendelian genetics (recall the Augustinian monk, Gregor Mendel 1822-1884) tells us that if the female can contribute only X while the male can contribute X or Y, the theoretical sex ratio at conception is 1:1.

Phenotypic Sex

"Phenotypic sex" encompasses all other than "genotypic sex."  Thus, phenotypic sex includes gonadal sex, genital sex and other aspects of physical appearance related to gender (including the so-called "secondary sex characteristics").  In humans, psychosexual determination is also a concern.  Evidence obtained from patients with phenotypic sex reversal suggests that psychological determination of sex is largely affected by environment and upbringing.  Our concern, here, is a clinical one.  The conclusion has been drawn that, in the event of "ambiguous genitalia" in a newborn infant, sexual reassignment is not precluded by genotypic sex.  A genotypic XX child raised as a male because of virilization of the genitalia, or a genotypic XY child raised as a female because of feminization of the genitalia can be perfectly normally socialized.  Clinical histories indicate that an XY female, raised as a female, perceives themselves as female, just as an XX male raised as a male would perceive themselves as male.  Beyond gonadal organogenesis, phenotype is determined to a great degree by exposure to the appropriate gonadal steroids.

PART B:  Gonadal Sex (Gonadal Organogenesis)

the Y chromosome

Is the Y chromosome the only chromosome involved in determination of male gonadal and genital sex or is Y assisted by, or under the regulation of the X chromosome and possibly autosomal chromosomes? What is H-Y antigen?  Does the Y chromosome do anything after sex is determined in the fetus?

Examination of abnormalities of sexual differentiation continue to suggest that X and Y chromosomes as well as the autosomes all carry genes influencing gonadal differentiation, causing the bipotential gonad to develop as a testis or as an ovary.

Eichwald and Silmser (1955) found that it was possible to graft skin from a female mouse to another female mouse.  Similarly, they could graft skin from one male mouse to another, or from a female to a male.  However, when they grafted skin from a male mouse (donor) to a female mouse (recipient), the donor skin (male) was rejected by the recipient (female).  The rejection of male tissue by a female was attributed to a specific histocompatibility protein, called H-Y antigen.  So male tissues produce a protein (H-Y antigen) which causes an immune response in females and leads to rejection of male donor tissue by females.  It has been recognized ever since, that this same H-Y antigen is responsible for development of some of the characteristics associated with males.  For example, H-Y antigen helps direct the formation of the testes in males.  Expression of the gene for production of H-Y antigen is one role of the Y-chromosome.  Expression of H-Y antigen is associated with the "heterogametic" sex in vertebrates, which is usually the male.  Males are not always heterogametic.  In birds, the females are heterogametic and the males are homogametic.

In mammals, females are homogametic (XX) and males are heterogametic (XY).

general information about H-Y antigen

H-Y antigen appears early in embryonic life (as early as the 8-16 cell morula stage).  H-Y antigen is a peptide with a molecular weight around 18000.  H-Y antigen has been detected on all cell membranes from normal X-Y males except from immature germ cells.  There are 2 H-Y antigen binding sites, one binds both H-Y antigen as well as other proteins with low affinity; a second receptor is very specific and binds only H-Y antigen.  This specific H-Y antigen receptor binds H-Y antigen with high affinity and is found only on gonadal cells.  OK, this tells us that the specific target tissue for H-Y antigen is the gonad!

H-Y antigen & testicular formation

H-Y antigen is at least partly responsible for testicular organogenesis from the bipotential fetal gonad.  Here we must note that both the gonads and genitalia are bipotential.  That is, they can become either male or female!

a) The primitive fetal gonad is bipotential - this means that the primitive gonad can develop into either a testis or an ovary.  The primitive gonad is made of a loose net of cells called the rete cells.  These rete cells develop in the area of the urogenital ridge (or gonadal ridge) seen in above picture.  The very primitive male gonads are referred to as the "rete testes" and primitive female ovaries as the "rete ovarii."  The terms rete testes and rete ovarii most specifically refer to the stromal tissue within the undifferentiated gonads of the genotypic male and genotypic female, respectively. "Rete" implies a network of cord-like tissue; the primitive gonads are, therefore, sometimes referred to as the primitive "sex cords."

Rete Testes	Rete Ovarii

b) There are 2 systems of ducts inside each fetus; one system has the ability to develop into the internal female reproductive tract (the paramesonephric duct or Müllerian duct system) and the other has the ability to develop into the internal male reproductive tract (the mesonephric duct or Wolffian duct system).

c) There is 1 set of primitive external genitalia which has the potential to follow either the male or female pattern of development.

H-Y antigen is secreted by Sertoli (sustenacular or nurse) cells of the very primitive gonad of a genotypic male fetus (46,XY).  These are the cells that eventually provide nutrients to the developing sperm cells.  Once secreted, H-Y antigen binds to H-Y receptors in the primitive male gonad and causes differentiation of the primitive gonad, from the rete testis to an identifiable testis by 6-7 weeks of gestation.  The most important structures of the testes are the seminiferous tubules.  The seminiferous tubules make up the bulk of the testes and, following puberty, will be the site of sperm production.

The lumen of the seminiferous tubules is the site of spermatogenesis. Outside of the seminiferous tubules lie primordial interstitial cells (Leydig cells).  Interstitial cells produce fetal androgens at this time, either autonomously or under the influence of human chorionic gonadotropin (hCG), a glycoprotein produced by syncytiotrophoblast cells of placenta.  In the absence of H-Y antigen and in the presence of 2 structurally normal X chromosomes, an ovary develops.

Ovary	Testes

In patients with testicular tissue who have XX karyotypes (eg. XX males and XX true hermaphrodites, who are extremely rare), despite the absence of a Y chromosome, H-Y antigen is detected in all patients tested (although it is usually at lower levels than observed in control 46,XY males). In addition, the gonad of the bovine freemartin (the "intersex" XX twin of a male fetus) tests positive for the presence of H-Y antigen.  Freemartinism in cattle is almost an invariable event in the case of co-gestation of a genotypic male and genotypic female. There are actually anastomoses between the placental circulations of the bovine fetuses which allow blood exchange of proteins which would not normally cross the placental barrier.  So it is suspected that in freemartinism, some of the H-Y antigen from the male fetus reached the circulation of the female twin and interfered with her normal pattern of development.  This is not normally a problem in cogestation of a human female fetus and male fetus.  Evidence does indicate, that even in the absence of a discrete Y chromosome or evidence of a Y to X or Y to autosome translocation, or insertion, the presence of some (even traces of) testicular tissue is invariably associated with a positive H-Y antigen test.

All of the evidence discussed previously in support of the testis-organizing function of H-Y antigen is indirect and circumstantial.  However, in vitro, H-Y antigen causes dissociated cells from the very primitive gonad of the male embryo to assemble into structures which are similar to seminiferous tubules.  If you simultaneously treat these same cells with H-Y antigen together with an antibody against H-Y antigen (to block the action of H-Y antigen), the cells will not form into structures similar to seminiferous tubules.  If you treat the very primitive, bipotential gonad from an XX fetus with H-Y antigen, it too will differentiate in a seminiferous tubule-like pattern, or what might be called a testicular pattern.  This tells us that H-Y antigen is a very important factor in the normal formation of the testes, but it does not tell us if the gene for H-Y antigen is the gene that actually causes formation of the testes.  This is kind of like saying, your car runs on gas but without a key, it won't start!  Well, in this example, testes formation proceeds with the help of H-Y antigen, but the processes involved are triggered by another gene; most likely by the SRY gene.

The SRY gene (sex determining region of the Y chromosome)

While H-Y antigen has long been suspected as the factor causing testicular differentiation, the "minimum gene requirement" for testicular organogenesis and the gene for H-Y antigen are not the same gene.  The site for expression of H-Y antigen is in the pericentromeric region of the Y chromosome, as well as on the short arm of the X chromosome and possibly on some autosomes.

The SRY gene is a single exon with 237 base pairs, located in the Yp 11.3 band of the Y chromosome.  It is the smallest part of the Y chromosome that is absolutely necessary for male sex determination (formation of the testes).  The SRY gene is located immediately next to the "pseudoautosomal region" at the distal end of the short arm of Y. The pseudoautosomal region is a region at the tip of the short arm of X and Y which is identical on the X and Y chromosome.  In mice, insertion of the SRY gene into the genome of embryonic female mice caused those mice to develop testes and external male genitalia.

One more interesting thing about the SRY gene.  It is present in the majority of "naturally occurring, sex-reversed" XY phenotypic females and is completely absent in "naturally occurring, sex reversed" XX phenotypic males.  It is now suspected that SRY exerts its effects by inhibiting a gene which prevents testes formation.

are other genes involved in testis formation?         

However, exchange of the pseudoautosomal regions can cause sex inversions (ie. sex inversions can be caused by exchange of material outside of the minimum region of the Y chromosome responsible for testicular organogenesis).  Once again, this tells us that the SRY gene is not the only thing that determines maleness; but the SRY gene is an absolute "must have" for functional testes.  At least two more candidates are the WT-1 tumor suppressor gene from chromosome 11 and the SF-1 steroidogenic factor gene, a nuclear receptor protein, found on chromosome 17.  SF-1 codes for hydroxylase enzymes required to manufacture steroids.  There also appears to be a sex reversal gene, which may be responsible for the occurrence of some 46,XY phenotypic females.  This gene is found on the short arm of the X chromosome and is known as the DAX-1 gene.  DAX-1 may prevent formation of the testes if the gene is duplicated on an X chromosome, or if genes normally suppressing DAX-1 are deleted from the X chromosome.  In other words, sex reversals could hypothetically result from insertions, deletions or translocations.  Some people suggest that DAX-1 may be functional in ovarian formation, but it is probably active very early on in formation of the primitive bipotential gonad.

"Seeding of the primitive gonad"

Ovarian organogenesis, encapsulation & the primordial follicle

In the gonad destined to be an ovary, lack of differentiation persists. At 77-84 days, long after formation of testes in a male fetus, the germ cells enter meiotic prophase to characterize the transition of oogonia into "primary oocytes."  By the way, at least one gene driving the entry of oogonia into meiosis has been identified.  This marks the onset of ovarian differentiation. When oogonia come into contact with rete ovarii, the rete ovarii lay down a layer of cells around the oogonia (encapsulation), a basement membrane forms immediately outside of the rete ovarii cells, and the oogonia are locked in prophase of meiosis I (primary oocytes).  We call the resulting structure a primordial follicle.

In summary, then, a primordial follicle consists of an oocyte halted in Prophase I (primary oocyte) and encapsulated in a single layer of rete ovarii.  Once the rete ovarii are encapsulated within a basement membrane, we refer to the rete ovarii as "granulosa cells."  It has been estimated that approximately 1700 primordial germ cells begin the migration from the yolk sac region to the urogenital ridge.   Millions of primary oocytes may be encapsulated in the developing fetal ovaries, by midgestation.  Again, follicles containing primary oocytes are referred to as primordial follicles.  Each primordial follicle contains a single primary ooctye.  Formation of the ovary, which started around mid-gestation, is relatively complete by 7 months of gestation.

PART C:  Genital Sex

By 7 weeks of gestation, the fetus is equipped with bipotential "primordia" of the male and female genital ducts (ie. the internal genitalia).  The Müllerian (paramesonephric) ducts, if allowed to persist, form the Fallopian tubes, uterus, cervix and upper third of the vagina. The Wolffian (mesonephric) ducts differentiate to form the epididymis, vas deferens, seminal vesicles and ejaculatory ducts of the male.  In the presence of a testis, the Müllerian (paramesonephric) ducts regress under the influence of Müllerian inhibiting hormone, a protein secreted by Sertoli cells. Differentiation of the Wolffian (mesonephric) duct system is stimulated by testosterone production by primitive Leydig cells.  Testosterone production by the primitive Leydig cells may be autonomous or may be stimulated by placental hCG.  Differentiation of male external genitalia occurs by 65-77 d gestation. Notice that, time-wise, this precedes differentiation of the bipotential gonad to the ovary at 77-84 days.  Dihydrotestosterone (DHT) is the major inducer of the male genitalia.  An X-linked gene controls expression of cytosolic androgen binding proteins.  Wolffian (mesonephros) inhibiting and Müllerian (paramesonephros) stimulating factors are produced in the female fetus to suppress development of the male internal genitalia and stimulate development of the female internal genitalia.  In the presence of maternal estrogens only, with an absence of androgens, the external genitalia develop towards the female pattern.

Ambiguous external genitalia

If the female fetus is exposed to excess androgens prior to 12 weeks of gestation, virilization of the genitalia may occur.  Historically, this occurred in a number of infants born to mothers who had taken "progestagen" analogs to prevent morning sickness.  Unfortunately, one  of the "progestagen" analogs (ie. a pharmaceutical agent) used was readily converted to androgens by the maternal adrenal cortex, exposing female fetuses to androgens and virilizing them.  A similar problem may occur if a pregnant female develops an androgen secreting adrenocortical tumor.  If androgen exposure occurs beyond 12 weeks of gestational age in the female fetus, there may be only partial virilization of the genitalia.  Likewise, insufficient androgen stimulation or response results in partial feminization of the male genitalia.  The most common occurrence of this condition is associated with a phenomenon referred to as tfm syndrome (testicular feminization), in which the male may or may not produce normal levels of androgens, but their androgen receptors do not respond adequately to androgens.  While androgen therapy may be used, it is not particularly successful because those with tfm syndrome are poor androgen responders.