Lata Murthy

Estrogen regulates expression of the jun family of protooncogenes in the uterus

Treatment of immature female rats with estradiol increases uterine levels of c-jun and jun-B mRNAs approx. 10-fold. This effect is specific for estrogenic steroids. The induction of jun transcripts is blocked by actinomycin D but not puromycin, suggesting that the hormonal effect is due at least in part to transcriptional activation. The hormone effect is rapid and peak levels of jun mRNAs are seen within 3 h after treatment. Inductions of jun and fos transcripts in the uterus by estradiol exhibit similar dose response curves (maximum responses at 4 micrograms/kg). Estradiol also elevates uterine levels of jun-D, and this induction is insensitive to puromycin. In vivo treatment with the phorbol ester TPA rapidly elevates uterine levels of fos, jun, and myc transcripts, indicating that expression of these protooncogenes is under non-estrogenic as well as estrogenic regulation in this target tissue. These results suggest that multiple members of the jun and fos protooncogene families may play a role in amplifying the uterine response to estrogens.

Association of mutations in the zona pellucida binding protein 1 (ZPBP1) gene with abnormal sperm head morphology in infertile men

Nearly 7% of men are afflicted by male infertility worldwide, and genetic factors are suspected to play a significant role in the majority of these patients. Although sperm morphology is an important parameter measured in the semen analysis, only a few genetic causes of teratozoospermia are currently known. The objective of this study was to define the association between alterations in the genes encoding the Golgi-associated PDZ- and coiled-coil motif containing protein (GOPC), the protein interacting with C kinase 1 (PICK1) and the acrosomal protein zona pellucida binding protein 1 (ZPBP1/sp38) with abnormal sperm head morphology in infertile men. Previous reports demonstrated that mice lacking Gopc, Pick1 and Zpbp1 are infertile due to abnormal head morphology. Herein, using our validated RNA-based method, we studied spermatozoal cDNA encoding the human GOPC, PICK1 and ZPBP1 genes in 381 teratozoospermic and 240 controls patients via direct sequencing. Among these genes, we identified missense and splicing mutations in the sperm cDNA encoding ZPBP1 in 3.9% (15/381) of men with abnormal sperm head morphology. These mutations were not observed in 240 matched controls and the dbSNP database (χ(2) = 9.3, P = 0.002). In contrast, statistically significant and functionally relevant mutations were not discovered in the GOPC and PICK1 genes. In our study ZPBP1 mutations are associated with abnormal sperm head morphology, defined according to strict criteria, resembling the mouse Zpbp1 null phenotype. We hypothesize that missense mutations exert a dominant-negative effect due to altered ZPBP1 protein folding and protein:protein interactions in the acrosome.

UBE2B mRNA alterations are associated with severe oligozoospermia in infertile men

Oligozoospermia (low sperm count) is a common semen deficiency. However, to date, few genetic defects have been identified to cause this condition. Moreover, even fewer molecular genetic diagnostic tests are available for patients with oligozoospermia in the andrology clinic. Based on animal and gene expression studies of oligozoospermia, several molecular pathways may be disrupted in post-meiotic spermatozoa. One of the disrupted pathways is protein ubiquitination and cell apoptosis. A critical protein involved in this pathway is the ubiquitin-conjugating enzyme 2B, UBE2B. Absence of Ube2b in male mice causes spermatogenic meiotic disruption with increased apoptosis, leading to infertility. To examine the association between messenger RNA defects in UBE2B and severe oligozoospermia (0.1-10 × 10(6) cells/ml), sequencing of sperm cDNA in 326 oligozoospermic patients and 421 normozoospermic men was performed. mRNA alterations in UBE2B were identified in sperm in 4.6% (15 out of 326) of the oligozoospermic patients, but not found in control men, suggesting strong association between mRNA defects and oligozoospermia (χ(2) = 19, P = 0.0001). Identified UBE2B alterations include nine splicing, four missense and two nonsense alterations. The follow-up screen of corresponding DNA regions did not reveal causative DNA mutations, suggesting a post-transcriptional nature of identified defects. None of these variants were reported in the dbSNP database, although other splicing abnormalities with low level of expression were present in 11 out of 421 (2.6%) controls. Our findings suggest that two distinct molecular mechanisms, mRNA editing and splicing processing, are disrupted in oligozoospermia. We speculate that the contribution of post-transcriptional mRNA defects to oligozoospermia could be greater than previously anticipated.

Sperm counts and sperm sex ratio in male infertility patients.

In recent years, investigators have noted a trend toward a declining proportion of male births in many industrialized nations. While men bear the sex-determining chromosome, the role of the female partner as it pertains to fertilization or miscarriage may also alter the gender ratio. We attempted to determine a mans secondary sex ratio (F1 generation) by directly examining the sex chromosomes of his sperm. We examined our male infertility clinic database for all men who had undergone a semen fluorescence in situ hybridization (FISH). Patient demographic and semen parameters were recorded. Chi-squared analysis was used to compare gender ratios (Y chromosomes/total chromosomes). Multivariable logistic regression was used to predict the odds of possessing a Y-bearing sperm after accounting for demographic and semen parameters. A total of 185 men underwent sperm FISH. For the entire cohort, the proportion of Y chromosome-bearing sperm was 51.5%. Men with less than five million motile sperm had a significantly lower proportion of Y chromosome-bearing sperm (50.8%) compared to men with higher sperm counts (51.6%; P=0.02). After multivariable adjustment, a higher sperm concentration, total motile sperm count and semen volume significantly increased the odds of having a Y chromosome-bearing sperm (P<0.01). As a mans sperm production declines, so does the proportion of Y chromosome-bearing sperm. Thus, a mans reproductive potential may predict his ability to sire male offspring.

The effect of estrogen on Sertoli cell function.

This study describes the use of adult rats as a model to determine whether chronic estrogen treatment irreversibly alters the capacity of Sertoli cells to secrete androgen binding protein (ABP). Twenty-five adult rats were implanted with silastic tubing containing 17 beta-estradiol. After 1 month of estradiol the epididymides had regressed to 47 per cent of the weight of the epididymides in a control group. The amount of ABP in control epididymides was 9.9 fmol./mg. protein, whereas no ABP was detectable in those from rats treated with estradiol for 1 month. Serum testosterone levels had been depressed by 90 per cent. Estradiol treatment for 8 months resulted in a 60 per cent lower body weight and a 91 per cent decrease in testicular weight compared to control animals. During this period the epididymides regressed significantly. When Sertoli cells were cultured from the testes of the estradiol implanted rats, in spite of the dramatic changes which had occurred in the testes, within 4 days the cultured cells synthesized ABP in response to FSH and testosterone. Furthermore, the direct addition of estrogen at a concentration of 200 ng./ml. to rat Sertoli cell cultures failed to depress ABP below control levels. Thus, although estradiol depresses ABP synthesis in vivo, probably through its depression of both gonadotrophin secretion and testosterone biosynthesis, it has no direct effect on the Sertoli cells, and its long-term inhibition of ABP synthesis by an indirect mechanism is reversible.

Correlation between simultaneous PSA and serum testosterone concentrations among eugonadal, untreated hypogonadal and hypogonadal men receiving testosterone replacement therapy

The primary objective of this study was to correlate simultaneous measures of prostate-specific antigen (PSA) and serum testosterone among large samples of eugonadal, untreated hypogonadal and hypogonadal men treated with testosterone replacement therapy (TRT). From 2001 to 2007, laboratory records were reviewed to identify men who underwent simultaneous measurement of PSA and serum testosterone levels. The data were stratified based on three groups of men: group 1 consisted of eugonadal men (T>300 ng per 100 ml) evaluated for BPH, reproductive failure or sexual dysfunction; group 2 consisted of untreated hypogonadal men (T<300 ng per 100 ml); and group 3 comprised symptomatic hypogonadal men receiving TRT. Correlations were found between PSA (total and free fractions) and total serum testosterone levels among the three groups. Group 1: eugonadal men (n=385 patients), mean PSA and serum testosterone were 1.60 ng ml−1 and 484 ng 100 ml, respectively. There was no significant correlation between PSA and total serum testosterone levels (r=−0.01, P=0.8). Group 2: untreated hypogonadal men (n=229 patients), mean PSA and serum testosterone were 1.49 ng ml−1 and 269 ng per 100 ml, respectively. There was no significant correlation between PSA and total serum testosterone levels (r=0.03, P=0.6). Group 3: hypogonadal men on TRT (n=229 patients and 994 individual samples analyzed) mean PSA and serum testosterone were 1.50 ng ml−1 and 555 ng per 100 ml, respectively. There was no significant correlation between PSA and serum testosterone levels (r=−0.005, P=0.9). Mean total serum testosterone levels were increased significantly (P<0.001) following treatment. Mean PSA levels did not increase in a statistically or clinically significant manner following TRT (mean PSA increase from baseline 0.05 ng ml−1, P=0.6). In conclusion, TRT does not appear to significantly influence serum PSA expression and no significant correlation was identified between PSA and serum testosterone among eugonadal, untreated hypogonadal and hypogonadal men receiving TRT.

Progesterone inhibits estrogen-induced increases in c-fos mRNA levels in the uterus.

Publisher Summary Progesterone decreases the induction of c-fos mRNA in the rodent uterus by estrogen. This inhibition raises the possibility that progesterone antagonism of estrogen-induced uterine growth is mediated in part by suppression of fos transcript levels. This effect of progesterone occurs in both the rat and mouse and it will be interesting to determine in future work if it occurs in other species. The dosage of progesterone that elicits a decrease in estrogen induced fos mRNA is in the range that produces other tissue responses to progesterone. These studies with other classes of steroids indicate that the effect of progesterone exhibits some specificity because neither an androgen nor a mineralocorticoid inhibits estrogen induction of c-fos mRNA. The results suggest that the most likely mechanism of the observed effect is a direct inhibition of transcription at the level of the c-fos gene. This inhibition could occur by one of several mechanisms. First, the progesterone receptor could bind to a regulatory region of the fos gene and prevent or alter the binding of estrogen receptor and or other transcription factors. Alternatively, the antagonistic effect of progesterone could be mediated by way of protein-protein interactions between its receptor and transcription factors required for estrogen induction of fos transcription.

Estrogen Regulation of Protooncogene Expression

Our basic approach to the problem of hormonal carcinogenesis has been to investigate the mechanisms by which physiologically relevant levels of estrogens regulate the growth and function of normal target tissues. This information is required to understand the mechanisms of carcinogenesis in hormone target tissues, the role of the hormone in these processes, and rational approaches to the prevent,ion and treatment of major human diseases such as prostate, endometrial, and breast cancer. The experimental system we have used for our studies is the immature rat uterus.

Phorbol esters inhibit estrogen-induced uterine DNA synthesis and increase apoptosis in uterine epithelium

Publisher Summary Numerous agents, including growth factors, protooncogenes, and transcription factors have been implicated in the regulation of uterine growth and function by estrogenic hormones. Another major signaling pathway involved in the cellular responses to many hormones, neurotransmitters, and growth factors involves protein phosphorylations catalyzed by protein kinase C. This chapter describes a study that used TPA to assess the effects of PKC activation on estradiol (E2)-induced proliferation of uterine cells in the immature rat model. TPA treatment decreased estrogen stimulated uterine DNA synthesis when administered 6 hr after the hormone. This suggests that a discrete event in the E2-induced growth program, which occured 6 hr after the process is initiated, was altered as the result of PKC activation. This effect of the phorbol ester is reversible and occurs in all major uterine cell types of the immature rat uterus. In addition to inhibiting DNA synthesis, TPA also affects apoptosis. The inhibitory effect of PKC occurs distal to receptor occupancy.

Estrogen Regulation of Uterine Proliferation: How Many ERRs Are Required?

Estrogen-stimulated endometrial proliferation is an important preparatory event for implantation and is thought to be initiated by estrogen receptor (E-R)-mediated changes in gene expression. Many laboratories have thus focused on the biochemical nature of E-R interactions and how this complex interacts with the consensus estrogen response element (ERE) originally identified in the vitellogenin genes of birds and amphibians.