When the Genetics of Gonadal Differentiation & Development Don’t Go According to Plan – Part 3 of Where Do Ovaries (and Testes) Come From?

File:Chromosome Y.svg

Map of the Humann Y-Chromosome

In the previous post, we talked about the genes that drive sex differentiation of the bi-potential embryonic gonad.  I only mentioned a relatively small number of genes, but they control the events at or near the beginning of the signaling cascades that give rise to the ovary or testis.  However, these genes are just the tip of the proverbial iceberg.

To put things into perspective, there are 266 genes that are turned on and 50 that are turned off in the mouse embryonic XY gonad during the window of sex determination (Beverdam & Koopman, 2006), and then 1086 genes are turned on in the subsequent cascade ( Nef et al., 2005).  The function of all of these genes has not yet been determined, but it was intersex conditions caused by the disregulation or mutation of some of these genes that initially led to their discovery.

So far in these posts, we have learned about where ovaries (and testes) come from and the genes driving their embryonic development.  In this post, we will consider situations when sex determination does not go according to plan, resulting in intersex conditions/disorders of sexual development (DSDs) with a basis in the gonad.

Let’s begin with SRY. Since 1905, when the X and Y sex chromosomes were first characterized, scientists assumed that there was a gene on the Y-chromosome that directed testis development.  In the 1950s, when chromosomal analyses improved, scientists realized that in individuals with no Y-chromosome, gonadal development occurred down the female path, regardless of the number of X-chromosomes the individual carried (reviewed in Ostrer, 2003). 

However, it was the individuals who were sex reversed with respect to their chromosomes that provided more information about the control of gonadal development.  With the advancements in karyotyping and chromosomal analyses, more information was derived about 46,XX individuals lacking a Y-chromosome who developed as males and 46,XY individuals who developed as females with ovarian gonadal dysgenesis. The question became, how could these cases of sex reversal occur if the Y-chromosome supposedly carried the gene responsible for testis development?

An answer was proposed with the characterization of non-homologous recombination in the 1960s.  During homologous recombination during meiosis, sequences of DNA are exchanged between paired sister chromosomes, a mechanism that contributes to biological diversity of the offspring.  Non-homologous recombination occurs when DNA sequences are exchanged between chromosomes that are not the same, such as between the X and Y chromosomes. 

As an FYI, there are other ways that chromosomes can incorrectly exchange genetic material.  There is translocation, where entire pieces of chromosomes are exchanged. 

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There is also insertion, where a piece of one chromosome is inappropriately spliced into another chromosome. These mechanisms might come into play with genetic anomalies not involving the sex hormones.

So, going back to the hypothesis for 46,XX gonadal sex reversal, the theory was that the gene (or genes) on the Y-chromosome that conferred male sex determination was being transferred to the X-chromosome during meiotic non-homologous recombination. Basically, a piece was removed from the Y-chromosome and joined to the X-chromosome during meiosis.  In this way, any gamete (egg or sperm) carrying the affected sex chromosomes would, upon conception, result in an offspring that would be chromosomally sex reversed. 

For example, an offspring carrying XX-chromosomes, one of which would be harboring the sex determining Y-chromosome gene(s), would be a genetic female with testis development.  Conversely, any offspring carrying the XY-chromosomes where the sex determining gene(s) on the Y-chromosome was missing because it had been transferred during meiotic non-homologous recombination to the X-chromosome, would develop as a female.  

 Several candidate genes for male sex determination were considered and excluded in the 1970s and 1980s, and then in 1990, the SRY gene was discovered (Gubbay et al, 1990; Sinclair et al. 1990).  However, even with the discovery of the SRY gene, only a subset of genetic sex reversal cases were explained.

Of individuals that have XY chromosomes but develop as females (male-to-female sex reversal), the absence of the SRY gene on the Y-chromosome can account for the genetic sex reversal in only 15% of these individuals.  Similarly, of individuals carrying XX chromosomes and develop as males (female-to-male sex reversal, also known as 46,XX testicular DSD) the presence of the SRY gene on one of the X-chromosomes of these individuals accounts for only 75% of the known cases (reviewed in Nef & Vassalli, 2009).  Similarly, SRY is present in only 10% of 46,XX individuals with ovotesticular DSD.  Therefore, the existence of other sex determining genes were hypothesized, some of which were identified and characterized, but there are more that have yet to be found.

Sex Determining Genes on the Sex Chromosomes

Before talking about genes involved in sex determination, let’s take a moment to look at the sex chromosomes themselves.  Why are these paired chromosomes so different when all other pairs in the human karyotype are twins?  Poloumienko (2004 and references therein) provides a nice little review on the subject: 

 The evolution of mammalian X and Y sex chromosomes is arguably one of the most intriguing biological processes. The sex chromosomes evolved from an ordinary pair of ancient autosomes (Ohno 1967). The Y chromosome was then progressively degraded because of drift and selection processes, mostly retaining genes responsible for male-specific functions (Charlesworth 1990; Waters et al. 2001; Lahn et al. 2001). Human sex chromosomes share two pseudoautosomal regions, PAR1 and PAR2, which pair and recombine in meiosis. They are located at the tips of the short and long arms and together contain only 13 active genes (Toder et al. 2000; Gianfrancesco et al. 2001; Charchar et al. 2003; Iwase et al. 2003). . . . The genes of the human nonrecombining region on the Y chromosome (NRY) can be divided into two categories: testis-specific genes and those that have X-linked homologs (Lahn et al. 2001). Most of the gene pairs of the latter category have retained a high degree of sequence identity and are ubiquitously expressed.

Maps of the X and Y Chromosomes

We have already talked about the SRY gene, the “Testis Determining Factor (TDF)” which is located on the Y-chromosome.  Although several genes on the Y-chromosome were initially thought to be the TDF (reviewed in Ostrer, 2003), none panned out and so, to date, there are no other sex determining genes identified on the Y-chromosome, although genes necessary for male fertility are known, such as the DAZ1-4 genes (deleted in azoospermia 1-4), also known as the region AZFc (azoospermia factor c), USP9Y (ubiquitin specific peptidase 9, Y-linked, also known as AZFa), and DDX3Y (DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked, also known as DBY and AZFa).  At one time, ZFY was thought to be the TDF, and currently, the function of its encoded zinc-finger protein is unknown.

While the Y-chromosome is one of the smallest in the human karyotype and carries relatively few genes (estimated at 70-200), the X-chromosome is significantly different.  The X is quite large, relatively speaking, making up approximately 5% of the total DNA in the human karyotype, and carries somewhere between 900 and 1400 genes. 

Before discussing X-linked genes involved in sex determination, I will briefly mention and provide links for summaries chromosomal aneuploidies that affect gonadal/reproductive development and/or fertility: 
Klinefelter Syndrome – 47,XXY, 48,XXXY, 49,XXXXY, 46,XY/47XXY (mosaicism)
Turner Syndrome – 45,XO, 45,XO/46,XX (mosaicism)
48,XXYY Syndrome
46,XX testicular disorder of sex development – sex reversed because of SRY translocation

Of mention are Triple X Syndrome (47,XXX) and 47,XYY Syndrome, both of which have associated developmental issues that do not involve the reproductive organs or fertility.

Now, going back to our discussion of sex determining genes on the sex chromosomes, one that is on the X-chromosome that is involved in gonadal determination and development is ATRX in the XH2 region.  Mutation of this gene results in Alpha Thalassemia X-Linked Mental Retardation Syndrome.  Associated with a host of developmental issues, some males with this syndrome also experience reproductive developmental effects such as gonadal dysgenesis or ambiguous genitalia  (McPherson et al., 1995; Reardon et al, 1995).

Another gene in the XH2 region is NR0B1 (nuclear receptor subfamily 0, group B, member 1), which encodes the orphan nuclear receptor DAX1 (dosage sensitive sex reversal adrenal hypoplasia congenital critical region of the X chromosome gene 1).  The function of DAX1 is key for the development of hormone-producing reproductive organs(gonads, hypothalamus and pituitary) and the adrenal glands. 

Duplication of this region of the X-chromosome, resulting in an extra copy of the NR0B1 gene, or a mutation in the promoter region of the gene, both contribute to an over-expression of DAX1 during embryonic development.  The extra DAX1 somehow interferes with testis determination, resulting in sex reversal (female development) with gonadal dysgenesis (also known as Swyer Syndrome) in affected 46,XY individuals.  In the opposite direction, deletion of the NR0B1 gene results in the condition called X-linked adrenal hypoplasia congentia, where a sub-set of the 46,XY males with this condition have reduced androgen production (hypogonadotropic hypogonadism) with associated effects on the reproductive system.  It’s these differential effects of the “dose” of DAX1, whether too much or not enough, that contributes to its name.

Sex Determining Genes on the Autosomal Chromosomes

A number of sex determining genes are not located on the X/Y sex chromosomes, but are, instead, located on non-sex chromosomes, called autosomal chromosomes.   In the human karyotype, there are 22 pairs of autosomal chromosomes and one pair of sex chromosomes.  (In the image below, the Y-chromosome is the single blue one at the top center and the X-chromosomes is the single red one at the right.)


The first autosomal gene that was characterized as a testis-determining gene was WT1 (Wilms’ Tumor Suppressor).  Located on Chromosome 11, WT1 encodes a transcription factor that is expressed both before and after differentiation of the bi-potential gonad to testis.  Alternative splicing results in 5 different isoforms that are involved in cancer cell metastasis, proliferation and death mechanisms.  Expressed in the embryonic kidney and gonads, mutation of WT1 results in development of Frasier disease and Denys-Drash Syndrome that include Wilms’ (kidney) tumor, a number of kidney and urogenital diseases as well as a number of different gonadal abnormalities including complete dysgenesis and 46,XY sex reversal (reviewed in Ostrer, 2003).  WT1 is a gene that is important early in gonadal development, at the point of the bi-potential gonad, and therefore for devleopment of both ovary and testis (reviewed in Kousta et al., 2010).

Another gene important early in embryonic development of the gonad encodes steroidogenic factor 1 (SF1), a transcription factor with a structure similar to steroid hormone receptors and important in steroid hormone production by the gonads and adrenal glands.  When it was first characterized in the early 1990s, SF1 was categorized as a nuclear orphan receptor because its ligand was unknown.  Approximately ten years after its initial identification, the ligand for SF1 was determined to be phospholipids such as sphingosine (Urs et al., 2006). 

SF1 is encoded by the NR5A1 gene (nuclear receptor subfamily 5, group A, member 1), which is located on Chromosome 9.  Mutations of this gene cause reproductive abnormalities, including gonadal dysgenesis/sex reversal in 46,XY individuals (also known as Swyer Syndrome), some of whom also experiencing adrenal insufficiency.  The range of gonadal and reproductive phenotypes may be due, in part, to the importance of SF1 in the regulation of other genes important in reproductive development and function, such as STAR (steroidogenic acute regulatory protein), CYP17A1 (17α-hydroxylase), CYP11A1 (cytochrome P-450 side-chain cleavage), LHB (luteinizing hormone β subunit), AMH (anti-Müllerian hormone), CYP19A1 (aromatase) and INHA (inhibin α subunit).  In addition, mutations in NR5A1 46,XX women have been linked to primary ovarian failure, also known as nonsyndromic ovarian insufficiency (Lourenço et al., 2009 and references therein).

The WNT4 gene (wingless-type MMTV integration site family, member 4), which was discussed in my previous post as being important in sex determination and development of the ovary, is located on Chromosome 1 and encodes a secreted protein that has dose-dependent effects similar to DAX1.  Also similar to DAX1, WNT4 protein regulates signaling pathways important during embryonic development of the Müllerian ducts, Leydig cells (the androgen-producing cells of the testis) and oogonia.  Whereas loss of WNT4 in 46,XX females results in a lack of development of Müllerian ducts and their derivatives and ovarian dysfunction, including overproduction of androgens, an extra copy of the WNT4 gene in 46,XY individuals causes incomplete sex reversal, with some underdeveloped Müllerian structures present and non-functional testes.

Also believed to be important for ovarian determination and development is FOXL2 (forkhead box L2), which was mentioned in my previous post.  The data so far show that although functional FOXL2 is required for proper ovarian development in mice and goats, it might not be as significant for determination of the human ovary.  Located on Chromosome 3 and expressed in both the embryonic ovary and eyelid, mutations of this gene in humans and loss of the transcription factor it encodes results in blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES), characterized by eyelid malformations (types I and II BPES) and premature ovarian failure in 46,XX females (type I BPES).    The gene is also known as BPESC1 (BPES candidate 1).  Although mutations in addition to the over 130 that are currently known for this gene might be found that induce human ovarian dysgenesis, the current thinking is that in humans, FOXL2, by itself, might not be an ovary determination gene as it is in goats and mice (reviewed in Nef & Vassalli, 2009 and Kousta et al., 2010).

One gene mentioned in the previous post that is definitely known to be an ovary-determining gene in humans is RSPO1 (R-spondin 1 homolog).  Located on Chromosome 1, mutation of RSPO1 in humans results in 46,XX sex reversal (testicular DSD) or ovotesticular DSD, accompanied by palmoplantar hyperkeratosis (thickening of the palms and soles), predisposition to squamous cell carcinoma of the skin, congenital bilateral corneal opacities, onychodystrophy (malformations and/or discolorations in the fingernails and/or toenails) and hearing impairment (reviewed in Kousta et al., 2010).

SOX9 (SRY-like HMG box protein 9) is also an autosomal sex-determining gene, and was also discussed in my previous as a testis-determining gene.  Located on Chromosome 17, loss of one SOX9 gene results in campomelic dysplasia, characterized by skeletal abnormalities and, in 75% of cases in 46,XY  individuals, sex reversal or ambiguous genitalia (Wagner et al., 1995).  In addition, 46,XX individuals who have an extra copy of the SOX9 gene have autosomal sex reversal (testicular DSD; Huang et al., 1999).  These effects of SOX9 are understandable, as it is immediately downstream of SRY in the male sex determination pathway (reviewed in DiNapoli & Capel, 2008).

DMRT1 (doublesex and mab-3 related transcription factor 1) is a sex-determining gene in invertebrates, might be the SRY-equivalent in birds (Koopman, 2009) and is potentially involved in cancer susceptibility (Kang et al., 2010).  Although deletions in the region on Chromosome 9 where DMRT1 is located have resulted in hypoplastic, undescended testes and ambiguous genitalia in 46,XY sex reversed individuals (Ogata et al., 1997; Flejter et al., 1998, Veitia et al., 1997), the jury is still out as to whether DMRT1 is a sex determining gene in humans.

Located on Chromosome 6, mutations in  TSPYL1 (TSPY-like 1; TSPY1 being a separate gene, testis specific protein, Y-linked 1, located on the Y-chromosome, important for spermatogenesis and involved in germ cell cancer) results in SIDDT, Sudden Infant Death with Dysgenesis of the Testes Syndrome.  First identified in an Amish family, SIDDT involves disregulation of autonomic and visceral nerves, usually resulting in death by 12 months of age because of cardio or respiratory failure, where affected 46,XY individuals have testicular dysgenesis with either ambiguous or female genitalia (Puffenberger et al., 2004). 

Lastly, the DHH gene (Desert hedgehog) encodes a signaling protein that is part of the Hedgehog family of developmental morphogens.  Located on Chromosome 12, mutations in the DHH gene have been found in 46,XY inviduals with sex reversal/complete gonadal dysgenesis (also known as Swyer Syndrome) and 45,XO/46,XY individuals with mixed gonadal dysgenesis, all in the absence of mutations in SRY, and sometimes associated with abnormal conductance of peripheral nerves (Canto et al., 2004; 2005).

It’s worth noting that a number of these genes encode transcription factors, which are proteins that bind to the DNA of other genes, potentially turning them on or off.  A mutation in a transcription factor, therefore, could be amplified in its effects, causing improper signaling of a number of other genes in the cascade.  For example, SF1 and WT1 can act synergistically with SOX9 to increase expression of the Müllerian Inhibiting Substance (MIS) gene, whereas DAX1 can act antagonistically (reviewed in Ostrer, 2003).  Therefore, mutation or over-expression of any of the genes mentioned could alter the way MIS is expressed, potentially resulting in failure of regression of female reproductive ducts in 46,XY individuals.

Adding to Our Knowledge
The understanding of human sex determination and development is constantly evolving.  Indeed, some of the genes mentioned above were only linked to gonadal development in the last decade.  Because of the complexity of sex determination and the lack of known genetic basis for a significant number of intersex/DSDs cases, coupled with the improved and rapid techniques for pinpointing and identifying genes, the list of genes known to be involved in the sex determination signalling cascades is likely to grow relatively quickly. 

Koopman et al., 1991

A number of these genes have been identified through studies with mice, a species used heavily in research because of our ability to manipulate the mouse genome.

Take, for instance, this photo on the right, which graced the cover of the May 9, 1991 issue of the scientific journal Nature.  (At least, that’s what I remember of it anyway.) 

What we see are two male mice, so judged by their genital-anal distance, which is greater in male versus female mice.  These mice, generated in Dr. Robin Lovell-Badge’s lab at the MRC in London (Koopman et al., 1991), were actually XX chromosomal females, sex reversed because they had been engineered to carry the murine Sry gene.  I was a graduate student when this paper came out, and it caused quite a stir and got a lot of international attention in the press, as it proved that SRY was the long-sought testis-determining gene.   Other sex determining genes have also been identified and/or characterized by using genetically engineered mice, either by introducing extra copies of a gene, such as with transgenic mice (like the Sry mice), or by removing a gene, which can be done with “knock-out” mice.

Whether through the study of the genomics of mice, humans or other species, we will likely see new information arise in the not too distant future regarding genetic control of sex determination and development.  As a prelude, “hints” for genes important in ovarian development already exist for follistatin, bone morphogenic protein 2 (BMP2), GATA4 and Fog2.

Incidence vs. Prevalence

It might seem fitting that today’s post comes at the end of Rare Disease Day in the USA.  The National Organization for Rare Disorders  (NORD) has teamed up with Rare Diseases Europe (EURORDIS) to bring awareness about the over 6000 rare or “orphan” diseases in the U.S. that get relatively little attention and funding for research.

A number of the syndromes and conditions mentioned in this post are considered rare diseases by NORD, such as Swyer syndrome, which has a number of genetic causes (mutations in SRYDHH, NR0B1, NR5A1 and others) and BPES.  NORD characterizes rare diseases as those that affect less than 200,000 people in the U.S.  At 307 million people currently in the U.S., that amounts to abaout 1 out of every 1535 people.   In Europe, EURORDIS characterizes a disease as rare if it affects less than 1 in 2000 people. 

However, these statistics are for the prevalence of these diseases and syndromes, rather than the incidence.  The prevalence tells us how many people at one time have a certain condition, disease, syndrome or diagnosis, such as the number of people in the U.S. who currently have a congenital heart defect (as an example).  The incidence tells us how often the condition, disease or syndrome arises within the population, such as the number of babies born with a congenital heart defect, which is approximately 8 out of every 1000 newborns.

I bring this up because when I initially wrote this post, I had quoted some incidence statistics for intersex conditions/DSDs with a basis in the gonad, but after reading about incidence and prevalence statistics for these conditions and the disagreements and debates around the numbers and the way they have been derived, I decided to leave that discussion aside and just focus on the genetics themselves.

Thinking About PCOS in Trans Men

You might be asking yourself, then, why bother with this discussion about the genetics of sex determination and development anyway? What does this topic have to do with PCOS in trans men, the eventual subject for these series of blog posts about the ovary? The answer has two parts.

First, as I mentioned in the first post in this series, my opinion was that in order to talk about PCOS or other ovarian malfunctions, a discussion regarding ovarian determination and development would provide a background for a future post on PCOS. It’s easier to understand what’s happening when things go wrong when we know what’s supposed to happen when things go right.

Second, we are beginning to understand that genes involved in embryonic ovarian determination and development can also play an important role in ovarian function, and perhaps PCOS, in the adult. Take, for example, NR5A1 which encodes SF1. Certain mutations in this gene have detrimental effects on embryonic gonadal development whereas others allow ovarian determination and development to procede but somehow cause premature ovarian failure in the adult female (Lourenço et al., 2009 and references therein). This concept may become important when we discuss the proper function of adult human ovaries and PCOS in trans men.

Wrap-Up
Having said that, I’m afraid that I won’t be able to make any more scientific posts until April, including any about PCOS in trans men.  I have to write a science-based book chapter by the end of March and so I won’t have time to work on both the book chapter and this blog, at least not scientifically speaking. I will still make non-scientific posts, and so for those of you who have been hoping for a break from the science, you’re in luck.

Above all, I appreciate all of you readers and commenters out there and would like to thank you for your interest in this blog.

 –ATM

References
Beverdam A & Koopman P, 2006.  Expression profiling of purified mouse gonadal somatic cells during the critical time window of sex determination reveals novel candidate genes for human sexual dysgenesis syndromes.  Hum Mol Genet 15:417-431

Canto P, Söderlund D, Reyes E and Méndez JP, 2004b. Mutations in the desert hedgehog (DHH) gene in patients with 46, XY complete pure gonadal dysgenesis. J Clin Endocrinol Metab 89:4480–4483.

Canto P, Vilchis F, Söderlund D, Reyes E, Méndez JP, 2005.  A heterozygous mutation in the desert hedgehog gene in patients with mixed gonadal dysgenesis.  Mol Hum Reprod 11:833-836

DiNapoli L, Capel B, 2008. SRY and the standoff in sex determination.  Mol Endocrinol 22:1-9

Flejter WL, Fergestad J, Gorski J, Varvill T, Chandrasekharappa S, 1998. A gene involved in XY sex reversal is located on chromosome 9, distal to marker D9S1779. Am J Hum Genet 63:794-802

Kang JU, Koo SH, Kwon KC, Park JW, 2010.  Frequent silence of chromosome 9p, homozygous DOCK8, DMRT1 and DMRT3 deletion at 9p24.3 in squamous cell carcinoma of the lung. Int J Oncol. 2010 37:327-335

Koopman P, 2009.  Sex determination: the power of DMRT1.  Trends Genet 25:479-481

Kousta E, Papathanasiou A, Skordis N, 2010.  Sex determination and disorders of sex development according to the revised nomenclature and classification in 46,XX individuals.  Hormones (Athens) 9:218-131.

Lourenço D, Brauner R, Lin L, et al., 2009.  Mutations in NR5A1 associated with ovarian insufficiency.  N Eng J Med 360:1200-1210

McPherson EW, Clemens MM, Gibbons RJ et al., 1995.  X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome: A new kindred with severe genital anomalies and mild hematologic expression.  Am J Med Genet 55:302-306

Nef S, Schaad O, Stallings NR, Cederroth CR et al., 2005.  Gene expression during sex determination reveals a robust female genetic program at the onset of ovarian development.  Dev Biol 287:361-377

Ogata T, Muroya K, Matsuo N, Hata J, Fukushima Y, Suzuki Y, 1997. Impaired male sex development in an infant with molecularly defined partial 9p monosomy: implication for a testis forming gene(s) on 9p. J Med Genet 34:331-334

Poloumienko A, 2004.  Cloning and comparative analysis of the bovine, porcine, and equine sex chromosome genes ZFX and ZFY.  Genome 47:74-83

Reardon W, Gibbons RJ, Winter RM et al., 1995.  Male pseudohermaphroditism in sibs with the alpha-thalassemia/mental retardation (ATR-X) syndrome.  Am J MEd Genet 55:285-287

Urs AN, Dammer E, Sewer MB, 2006.  Sphingosine regulates the transcription of CYP17 by binding to steroidogenic factor-1.  Endocrinol 147:5249-5258

Veitia R, Nunes M, Brauner R, Doco-Fenzy M, Joanny-Flinois O, Jaubert F, Lortat-Jacob S, Fellous M, McElreavey K, 1997. Deletions of distal 9p associated with 46,XY male to female sex reversal: definition of the breakpoints at 9p23.3-p24.1. Genomics 41:271-274 

 

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6 Responses to When the Genetics of Gonadal Differentiation & Development Don’t Go According to Plan – Part 3 of Where Do Ovaries (and Testes) Come From?

  1. j says:

    Not being a science person, I’m going to read this many times. But it’s very, very informative. As always.
    Thank you for the trouble you have taken to write this. It must have taken you many hours. Thank you.

  2. J says:

    To a man who has to live with pcos that is threatening his transition process it’s more than a blog post… It is empowerment. Even if he has not the education to understand, he can take printouts of this to his doctors who would be grateful to read your theory. Endos love this kind of involvement. They need these kind of inputs. Thank god you are doing this! Bless you!

  3. Mac says:

    Thanks for a great post. Plenty of information to keep me busy for a couple of months, maybe longer! I for one appreciate your posts and your time.

    Mac

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