Amplification of cDNA by polymerase chain reaction (PCR) was performed using an annealing temperature and number of cycles optimized for each gene. For each PCR reaction, the number of cycles used was optimized so that the amplification process was carried out within the exponential (linear) range as demonstrated in Figure 1. To perform a semi-quantitative analysis of samples, serial dilutions of cDNA were subjected to increasing PCR cycles in order to define the linear amplification range for each primer set.
Semiquantitative Reverse Transcription-Polymerase Chain Reaction
QIAGEN RNAeasy kit (QIAGEN, Venlo, The Netherlands) was used to extract mRNA according to the manufacturer’s instructions. Reverse transcription was carried out with 2 |xg of sample in 20 |xl of reaction mixture containing 10 mM each of dATP dCTP, dGTP, and dTTP; 20 pmol oligo (dT); 40 u/^l of ribonuclease inhibitor; 10 u/^l of avian myeloblastosis virus-reverse transcriptase; and 5X AMV-RT buffer (42°C, 60 min; 95°C, 5 min: Eppendorf Mastercycler Gradient, Brinkmann, West-bury, NY). Primers that specifically amplified each of these genes were designed and tested; four pairs of primers were used for subsequent HOX amplification.
Cells were plated in plastic flasks (75 cm2, Falcon, Franklin Lakes, NJ), maintained at 37°C in a humidified atmosphere (5% CO2 in air), and grown to confluence. The stromal cells were passed by trypsinization and plated in culture dishes (100-mm diameter) and were allowed to replicate to confluence. Immunocytochemical analysis of endometrial cells was conducted after the first passage.
Endometrium was collected from 10 normal-cycling reproductive-age women by endometrial biopsy with informed consent, under an approved Human Investigations Committee protocol. Tissue was immediately frozen in liquid nitrogen and stored at -80°C. Some of the tissue was fixed in formalin for histological examination or in paraformaldehyde for in situ hybridization. Menstrual-cycle dating was determined by menstrual history and confirmed by histological examination by using the criteria of Noyes et al.
Targeted disruption of both Hoxd9 and Hoxd10 does not alter fertility in female mice and does not result in abnormalities on uterine structure or position. The HOXD10 gene has been shown to be expressed in the human uterus. The expression of HOXD10 in tumors of the uterus, but not ovary or cervix, suggests that it too may also play a role in specifying human uterine identity. Hoxd11 is expressed anteriorly from Hoxa11 or Hoxc11. No genito-urinary abnormality could be detected in Hoxd11 (-/-) female mice; loss-of-function females were fertile.
The sixteen 5′ Hox genes belonging to the paralogous groups 9-13 all show DNA sequence similarities to the Drosophila Abdominal-B (Abd-B) gene, which specifies the identity of the most posterior segments of the larval and adult fly. Among HOX genes, members of the HOXC and HOXD clusters, and in particular the Abd-B-related 5′ genes of these linkage groups, are the least well characterized. In the developing genitalia, prominent expression of both Hoxc10 and Hoxc11 are observed in the posterior urogenital sinus, which gives rise to urethra and vagina. Later, their expression is seen at high levels in parameso-nephric duct and in the genital tubercle.
Two genes of the Hoxa cluster, Hoxa10 and Hoxa11, are expressed in localized areas of the paramesonephric duct destined to become the uterus or the lower uterine segment and cervix, respectively. Hoxa10 and Hoxa11 gene expression is necessary for endometrial development, allowing uterine receptivity to implantation. Female Hoxa10 (-/-) or Hoxa11 (-/-) homozygous mutant mice have uterine factor infertility. Although these mice ovulate normally, they are unable to support implantation. Embryos from Hoxa10 (-/-) mice successfully implant in pseudopregnant wild-type surrogates; however, wild-type embryos do not implant in Hoxa10- or Hoxa11-deficient uterus.
Uterine endometrium is an extremely dynamic tissue, undergoing sequential developmental changes in preparation for implantation during each estrous cycle. The successful implantation of the blastocyst requires successful development of uterine endometrial receptivity. In many ways, cyclic endometrial development in the adult can be considered analogous to embryonic development. Many of the genes traditionally thought of as regulators of embryonic development are also used to regulate endometrial development. Hox genes, essential regulators of morphogenesis and tissue differentiation in the embryo, are also essential for endometrial development and for endometrial receptivity.