David T. MacLaughlin

Early follicular serum müllerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles

OBJECTIVE To test the hypothesis that the concentration of early follicular phase serum müllerian-inhibiting substance (MIS) is associated with ovarian response in women undergoing ovulation induction in preparation for assisted reproductive technology (ART). DESIGN Retrospective analysis of frozen day 3 serum samples. SETTING Academic ART program. PATIENT(S) One sample of frozen day 3 serum from women with < or = 6 retrieved oocytes (n = 28) compared with women with > or = 11 oocytes retrieved (n = 79) in preparation for IVF. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Comparison of day 3 serum MIS levels between two groups of women. Other comparisons included maximum serum E(2) concentrations, number of retrieved oocytes, and percentage of mature oocytes between groups. RESULT(S) Mean serum MIS concentrations were 1.0 +/- 0.4 ng/mL compared with 2.5 +/- 0.3 ng/mL, or more than a 2.5-fold greater serum concentration of MIS in the group with > or = 11 oocytes retrieved compared with in the group with < or = 6 retrieved oocytes. CONCLUSION(S) These data demonstrate an association between early follicular phase serum MIS and the number of retrieved oocytes. Higher day 3 serum MIS concentrations were associated with greater number of retrieved oocytes.

Mutations in LRP2 , which encodes the multiligand receptor megalin, cause Donnai-Barrow and facio-oculo-acoustico-renal syndromes

Donnai-Barrow syndrome is associated with agenesis of the corpus callosum, congenital diaphragmatic hernia, facial dysmorphology, ocular anomalies, sensorineural hearing loss and developmental delay. By studying multiplex families, we mapped this disorder to chromosome 2q23.3–31.1 and identified LRP2 mutations in six families with Donnai-Barrow syndrome and one family with facio-oculo-acoustico-renal syndrome. LRP2 encodes megalin, a multiligand uptake receptor that regulates levels of diverse circulating compounds. This work implicates a pathway with potential pharmacological therapeutic targets.

A time-less function for mouse Timeless

The timeless (tim) gene is essential for circadian clock function in Drosophila melanogaster. A putative mouse homolog, mTimeless (mTim), has been difficult to place in the circadian clock of mammals. Here we show that mTim is essential for embryonic development, but does not have substantiated circadian function.

Measurements of serum mullerian inhibiting substance in the evaluation of children with nonpalpable gonads

BACKGROUND Müllerian inhibiting substance, produced constitutively by the prepubertal testes, promotes involution of the müllerian ducts during normal male sexual differentiation. In children with virilization and nonpalpable gonads, only those with testicular tissue should have detectable serum concentrations of müllerian inhibiting substance. METHODS We measured serum mullerian inhibiting substance in 65 children with virilization at birth and nonpalpable gonads (age at diagnosis, 2 days to 11 years) and serum testosterone in 54 of them either after the administration of human chorionic gonadotropin or during the physiologic rise in testosterone that occurs in normal infants. RESULTS The mean (+/-SD) serum mullerian inhibiting substance concentration in the 17 children with no testicular tissue was 0.7+/-0.5 ng per milliliter, as compared with 37.5+/-39.6 ng per milliliter in the 48 children with testes (P<0.001). In the latter group, the mean values in the 14 children with abnormal testes and the 34 with normal testes were 11.5+/-11.8 and 48.2+/-42.1 ng per milliliter, respectively (P< 0.001). The sensitivity and specificity of the serum müllerian inhibiting substance assay for detecting the absence of testicular tissue were 92 percent and 98 percent, respectively, as compared with 69 percent and 83 percent for the measurement of serum testosterone. Furthermore, measurement of serum mullerian inhibiting substance was more sensitive than serum testosterone measurement for the identification of children with abnormal testes (67 percent vs. 25 percent), whereas the specificity of the two tests was similar. CONCLUSIONS Measurements of serum mullerian inhibiting substance can be used to determine testicular status in prepubertal children with nonpalpable gonads, thus differentiating anorchia from undescended testes in boys with bilateral cryptorchidism and serving as a measure of testicular integrity in children with intersexual anomalies.

Mullerian-Inhibiting Substance

During the development of the male and female reproductive tracts, the Mul-lerian duct gives rise to the uterus, Fallopian tubes, and part of the vagina and the Wolffian duct gives rise to the epididymis, vas deferens, and seminal vesicles. The two ducts develop in the early embryonic stages of both sexes. Regression of one or the other duct occurs as a consequence of gonadal dif-ferentiation. In the female, Wolffian duct regression occurs passively due to the lack of testosterone, while, in the male, regression of the Mullerian duct is an active process controlled by a testicular factor called Mullerian-inhibiting substance (MIS) (Jost 1946a,b, 1947a,b). A critical step in characterizing MIS was the development of an organ culture assay where urogenital ridges from 14 1/2-day-old fetal rats were excised and cultured in vitro and then evaluated histologically for regression of the duct (Picon 1969). Based on this assay, MIS was identified as a protein produced by Sertoli cells of the fetal (Blanchard and Josso 1974) and newborn testis (Donahoe et al. 1977a) and subsequently purified to near homogeneity (Picard and Josso 1984; Budzik et al. 1985). The purified protein, a 140-kDa glycoprotein, is composed of two identical 70-kDa subunits.

Specific estradiol binding in schwannomas, meningiomas, and neurofibromas.

The cytoplasmic fractions of schwannomas (acoustic neuromas), meningiomas, and neurofibromas were assayed for the presence of estrogen receptors. Specific estradiol binding was detected in 7 of 16 schwannomas, 7 of 10 meningiomas, and 1 of 6 neurofibromas. A nontumorous vestibular nerve was also studied and showed no estradiol binding. In the tumors, the concentration of the estradiol binding sites as estimated by saturation binding analysis covered a wide range of values (21 to 2430 fmol/g of tumor) but, overall, meningiomas contained the highest amount of estradiol binder. A Scatchard plot analysis of one of the schwannoma specimens demonstrated high affinity estradiol binding (Ka = 1.695 X 10(10) M-1). Although there were more females than males in each tumor category, the overall incidence of estradiol binding was similar in males (5 of 11, 45%) and in females (10 of 21, 48%). In 5 cases, progestin binding was also measured and was detected in two meningiomas (both from female patients); one meningioma and two neurofibromas showed no progestin binding. A discussion is presented of the possible role of estradiol in the pathogenesis or modulation of meningeal and Schwann cell tumors as well as in the genetic disorder neurofibromatosis.

Müllerian-inhibiting substance type II receptor expression and function in purified rat Leydig cells.

Mullerian-inhibiting substance (MIS), a gonadal hormone in the transforming growth factor-β superfamily, induces Mullerian duct involution during male sexual differentiation. Mice with null mutations of the MIS ligand or receptor develop Leydig cell hyperplasia and neoplasia in addition to retained Mullerian ducts, whereas MIS-overexpressing transgenic mice have decreased testosterone concentrations and Leydig cell numbers. We hypothesized that MIS directly modulates Leydig cell proliferation and differentiated function in the maturing testis. Therefore, highly purified rat Leydig and Sertoli cells were isolated to examine cell-specific expression, binding, and function of the MIS type II receptor. These studies revealed that this receptor is expressed abundantly in progenitor (21-day) and immature (35-day) Leydig cells as well as in Sertoli cells. Prepubertal progenitor Leydig cells exhibit high affinity (Kd = 15 nm), saturable binding of MIS. No binding, however, is detected with either peripubertal imm...

MIS/AMH in the assessment of cryptorchidism and intersex conditions.

Mullerian inhibiting substance (MIS), also known as anti-Mullerian hormone (AMH), causes Mullerian duct involution during male sexual differentiation and also has a postnatal regulatory role in the gonads. Serum MIS/AMH has a gonad specific pattern of expression and its concentrations are sexually dimorphic in children; hence measurement of serum MIS/AMH helps in the evaluation of children with gonadal disorders. In boys with cryptorchidism (non-palpable gonads), serum MIS/AMH correlates with testicular tissue. A measurable value is predictive of undescended testes while an undetectable value is highly suggestive of anorchia. In minimally virilized phenotypic females, MIS/AMH helps differentiate between gonadal and non-gonadal causes of virilization. In children with intersex conditions, MIS/AMH values assist differential diagnosis: a value above the normal female range is predictive of testicular tissue, while an undetectable value is suggestive of absent testicular tissue. Thus, MIS/AMH is useful for delineating gonadal pathology and facilitates the differential diagnosis and management of children with diverse gonadal disorders.

Human ovarian cancer stem/progenitor cells are stimulated by doxorubicin but inhibited by Mullerian inhibiting substance

Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics. We have isolated from multiple ovarian cancer cell lines an ovarian cancer stem cell-enriched population marked by CD44, CD24, and Epcam (3+) and by negative selection for Ecadherin (Ecad−) that comprises less than 1% of cancer cells and has increased colony formation and shorter tumor-free intervals in vivo after limiting dilution. Surprisingly, these cells are not only resistant to chemotherapeutics such as doxorubicin, but also are stimulated by it, as evidenced by the significantly increased number of colonies in treated 3+Ecad− cells. Similarly, proliferation of the 3+Ecad− cells in monolayer increased with treatment, by either doxorubicin or cisplatin, compared with the unseparated or cancer stem cell-depleted 3−Ecad+ cells. However, these cells are sensitive to Mullerian inhibiting substance (MIS), which decreased colony formation. MIS inhibits ovarian cancer cells by inducing G1 arrest of the 3+Ecad− subpopulation through the induction of cyclin-dependent kinase inhibitors. 3+Ecad− cells selectively expressed LIN28, which colocalized by immunofluorescence with the 3+ cancer stem cell markers in the human ovarian carcinoma cell line, OVCAR-5, and is also highly expressed in transgenic murine models of ovarian cancer and in other human ovarian cancer cell lines. These results suggest that chemotherapeutics may be stimulative to cancer stem cells and that selective inhibition of these cells by treating with MIS or targeting LIN28 should be considered in the development of therapeutics.

KLE: a cell line with defective estrogen receptor derived from undifferentiated endometrial cancer.

KLE is a cell line derived from a poorly differentiated endometrial carcinoma that is aneuploid with chromosome numbers ranging from 51 to 66 and 6-8 marker chromosomes demonstrated by G banding. Tumors harvested from five of five nude mice bearing an inoculum for more than a month resemble the original specimen, and electron microscopy shows microvilli, many junctional processes, glycogenation, and a prominent nucleolonema. The cell cytosol contains a specific binder for estradiol, but there is no estrogen receptor in the nucleus and in a study reported elsewhere (Raam et al., Breast Cancer Res. Treat. 2, 277 (1982) ) translocation to the nucleus fails to occur. The enzyme phenotype of this cell is human, non-HeLa.