Chromothripsis orchestrates leukemic transformation in blast phase MPN through targetable amplification of DYRK1A.

Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a common event in cancer. Whether chromothripsis might constitute an actionable molecular event amenable to therapeutic targeting remains an open question. We describe recurrent chromothripsis of chromosome 21 in a subset of patients in blast phase of a myeloproliferative neoplasm (BP-MPN), which alongside other structural variants leads to amplification of a region of chromosome 21 in ∼25% of patients ('chr21amp'). We report that chr21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. The chr21amp event is highly clonal and present throughout the hematopoietic hierarchy. DYRK1A , a serine threonine kinase and transcription factor, is the only gene in the 2.7Mb minimally amplified region which showed both increased expression and chromatin accessibility compared to non-chr21amp BP-MPN controls. We demonstrate that DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development, including DNA repair, STAT signalling and BCL2 overexpression. DYRK1A is essential for BP-MPN cell proliferation in vitro and in vivo , and DYRK1A inhibition synergises with BCL2 targeting to induce BP-MPN cell apoptosis. Collectively, these findings define the chr21amp event as a prognostic biomarker in BP-MPN and link chromothripsis to a druggable target.

Craniospinal Irradiation in Medulloblastoma using High Energy Medical Linear Accelerator: an Innovative Approach to Planning Technique.

BACKGROUND: Craniospinal irradiation (CSI) of medulloblastoma poses technological challenges due to the involvement of large treatment volume. Commonly, the whole treatment length is covered with two different isocentric plans in which the junction is shifted after every five fractions to overcome the possibility of hot and cold spot. OBJECTIVE: This study aims to evaluate dosimetrically and clinically the innovative planning technique for the CSI which doesn't need re-planning and re-setup of patients after every five fractions. MATERIAL AND METHODS: Computed tomography was done for fifteen (ten children and five adults) patients diagnosed with medulloblastoma. Treatment planning for 36 Gray (Gy) in 20 fractions (#) at the rate of 1.8Gy/# was done on the treatment planning system. A single plan for children was created with two bilateral fields of 6 Mega Voltage (MV) energy for cranium and one posterior field of 6 MV for spinal cord (C1-S2). Two plans for adult patients were created, first plan was with two bilateral fields of 6 MV for cranium and two posterior oblique fields of 6 MV for cervical and the part of thoracic spinal cord (up to T8-T9). The second plan was with a single posterior field of 15 MV covering remaining thoracic (T8-T9 to T12), lumbar and sacrum (up to lower border of S2) spine. After careful evaluation of all the plans, treatment was delivered; acute toxicities were recorded. RESULTS: 95% of prescribed dose was received by more than 95% of planning target volume in all the plans with the acceptable hot spot and good homogeneity index. All the patients reported common radiation induced acute toxicities (headache, vomiting, weakness) during radiotherapy. CONCLUSION: The new planning technique for CSI has acceptable dosimetric and acute clinical possibilities; therefore it can be used for CSI for improved homogeneous dose delivery.

Intraluminal brachytherapy boost following external beam radiotherapy with concurrent chemotherapy of oesophagus carcinoma: Results of a prospective observational study.

PURPOSE: The main objective of our study is to evaluate response and toxicity profile in patients receiving external beam radiotherapy with concurrent chemotherapy followed by intraluminal brachytherapy boost for a carcinoma of the oesophagus. MATERIAL AND METHODS: Twenty patients with biopsy-proven carcinoma of the oesophagus received external beam radiotherapy (50Gy in 25 fractions) with concurrent chemotherapy (cisplatin: 40mg/m2). After a gap of two to three weeks, intraluminal brachytherapy (10Gy in two fractions each 1 week apart by a high dose rate 60Co source) was given. Response was evaluated at 1 month and at 1 year of completion of treatment. In addition, acute and chronic toxicity was evaluated at 1 month and 6 months of treatment. RESULTS: Complete response were seen in 80% of patients and partial response in 20% at 1 month. Moreover, there were 65% complete response, 10% local recurrences, 15% patients showed local control with distant metastasis and 10% patients died at 1 year. Grade 1, grade 2 and grade 3 oesophagitis were seen in 10%, 70% and 20% of patients respectively. Stricture was seen in 40% of patients and fistula in 10% of patients. There was no spinal cord, cardiac and nephrotoxicity found. CONCLUSIONS: With the concept that high tumoricidal dose for adequate tumor control achieved by intraluminal brachytherapy as a mean of dose escalation, while sparing surrounding normal tissue and potentially improving therapeutic ratio, external beam radiotherapy followed by intraluminal brachytherapy could be a better choice for oesophagus carcinoma.

Study of anemia in nondialysis dependent chronic kidney disease with special reference to serum hepcidin.

We studied the role of serum hepcidin in anemia of chronic kidney disease (CKD) in a hospital-based cross-sectional study. Serum hepcidin, ferritin, and high-sensitivity C-reactive protein (hsCRP) levels were evaluated in patients of CKD. Hepcidin levels were increased in patients as compared to healthy adults. Hepcidin levels increased as CKD progressed through stage 3-5 (P trend = 0.015) but did not correlate with estimated glomerular filtration rate. Hepcidin correlated positively with ferritin (P < 0.0001) and transferrin saturation (TSAT) (P = 0.0217) and negatively with erythropoietin (EPO) levels (P = 0.0258) but did not correlate with either hsCRP or estimated glomerular filtration rate. Iron status influenced hepcidin levels of patients. Patients were divided according to iron status on the basis of TSAT and serum ferritin levels. We observed that while absolute iron deficiency (transferrin saturation <20%, ferritin <40 ng/ml) is associated with downregulation of hepcidin, hepcidin is elevated in other two categories of CKD patients (P = 0.0039). Iron status of patients also influenced interaction between hepcidin and hemoglobin (Hb). Hepcidin correlated negatively with Hb in patients with sufficient iron status (r = -0.7452, P < 0.0001) but nearly correlated positively with Hb in patients with absolute iron deficiency (r = 0.9428, P = 0.0572). Almost similar association persisted when cutoff value for serum ferritin was raised to 100 ng/ml as per NKF/KDOQI 2006 clinical practice guidelines except that no association was observed in absolute iron deficiency category. Cutoff value for hepcidin for differentiating absolute iron deficiency from other categories in our study population is ≤ 34 ng/ml (area under curve = 0.836, P < 0.0001). In conclusion, serum hepcidin level is increased in nondialysis CKD patients as compared to healthy adults possibly due to associated inflammation and decreased renal clearance. Furthermore, iron status modifies hepcidin level and its association with Hb. Raised hepcidin can predict the need for parenteral iron therapy and need for higher dose of recombinant human EPO to overcome iron-restricted erythropoiesis.

Exposure of neonatal rats to anti-androgens induces penile mal-developments and infertility comparable to those induced by oestrogens.

We previously reported that oestrogen exposure in neonatal rats induced permanent infertility and malformed penis characterized by fat accumulation, which replaced most of the smooth muscle cells and cavernous spaces in the body of the penis, structures essential for erection. The objective of this study was to determine if reduced androgen production/action in the neonatal period, in the absence of exogenous oestrogen exposure, induces penile deformities similar to those caused by oestrogen. Male rats were treated from postnatal days 1-6 with GnRH antagonist antide (A, 10 mg/kg) or androgen receptor (AR) antagonist flutamide (F, 50 mg/kg) or F + A, with or without AR agonist dihydrotestosterone (DHT, 20 mg/kg). For comparison, pups received diethylstilbestrol (DES, 0.1 mg/kg), with or without DHT. Tissues were collected at ages 7 and 12 days and at adulthood. Flutamide alone decreased penile length and weight significantly (p < 0.05), but it caused neither fat accumulation, nor affected fertility (80% vs. 87% in controls). Antide alone reduced penile length and weight significantly, and induced fat accumulation in 4/11 rats and infertility in 13/14 rats. Conversely, all 11 F + A-treated rats, similar to all nine DES-treated rats, had fat accumulation and loss of smooth muscle cells and cavernous spaces in the body of the penis and were infertile. In addition, reductions in penile length and weight were higher than in rats treated with F or A alone. DHT co-administration mitigated penile deformities in the DES group, but did not in the F + A group. Testicular testosterone was reduced by 70-95% at 7 or 12 days of age in all treated groups, except in the F group, which had threefold higher testosterone than controls. Collectively, data unequivocally show that reduced androgen production/action in the neonatal period, in the absence of oestrogen exposure, induces permanent infertility and malformed penis similar to that caused by oestrogen.

Estrogen-induced developmental disorders of the rat penis involve both estrogen receptor (ESR)- and androgen receptor (AR)-mediated pathways.

This study tested the hypothesis that the estrogen receptor (ESR) pathway, androgen receptor (AR) pathway, or both mediate estrogen-induced developmental penile disorders. Rat pups received diethylstilbestrol (DES), with or without the ESR antagonist ICI 182,780 (ICI) or the AR agonist dihydrotestosterone (DHT) or testosterone (T), from Postnatal Days 1 to 6. Testicular T concentration, penile morphology and morphometry, and/or fertility was determined at age 7, 28, or 150 days. DES treatment alone caused 90% reduction in the neonatal intratesticular T surge; this reduction was prevented by ICI coadministration, but not by DHT or T coadministration. Unlike the T surge, coadministration of ICI and coadministration of DHT or T mitigated penile deformities and loss of fertility. Generally, ICI, DHT, or T treatment alone did not alter penile morphology; however, fertility was 20% that of controls in ICI-treated rats vs. 70%-90% in DHT- or T-treated rats. The lower fertility in the rats treated with ICI alone could be due to altered sexual behavior, as these males did not deposit vaginal plugs. In conclusion, observations that both an ESR antagonist and AR agonists prevent penile deformities and infertility suggest that both pathways are involved in estrogen-induced penile disorders. Observations that coadministration of ICI, but not DHT or T, prevents the DES-induced reduction in the neonatal T surge suggest that, although ICI exerts its mitigating effect both at the level of Leydig cells and penile stromal cells, DHT and T do so only at the level of stromal cells.

Role of estrogen in induction of penile dysmorphogenesis: a review.

In this review, we report permanent dysmorphogenesis of the penis and loss of fertility in adult rats treated neonatally with estrogen. Specifically, we report replacement of smooth muscle cells and cavernous spaces by fat cells in the corpus cavernosum penis, but not in the adjoining corpus spongiosum. Induction of these novel, region-specific phenotypes is dose-dependent, requires a critical window of exposure and associated with decreased testosterone and up-regulation of estrogen receptor alpha (ER alpha). The resistance of ER alpha knockout mice to develop these abnormalities implies an unequivocal role for ER alpha in mediating maldevelopment of the penis. Additionally, the prevention of estrogen-inducible penile abnormalities by ER antagonist ICI 182 780 implies that a functional ER-mediated pathway is essential for inducing penile abnormalities. Likewise, the ability of testosterone or dihydrotestosterone to negate these abnormalities suggests a role for an androgen receptor (AR)-mediated pathway. Taken together, these observations led us to hypothesize that neonatal estrogen exposure, via an ER-mediated pathway (direct action) or an AR-mediated pathway (indirect action through decreased testosterone) or both pathways, up-regulates ER alpha expression in stromal cells of the penis, which are then reprogrammed such that their differentiation into smooth muscle cells is inhibited and their differentiation into adipocytes is stimulated.

Estrogen receptor alpha mediates estrogen-inducible abnormalities in the developing penis.

Previously, we reported an association between estrogen receptor-alpha (ERalpha) upregulation and detrimental effects of neonatal diethylstilbestrol (DES) exposure in the rat penis. The objective of this study was to employ the ERalpha knockout (ERalphaKO) mouse model to test the hypothesis that ERalpha mediates DES effects in the developing penis. ERalphaKO and wild-type C57BL/6 mice received oil or DES at a dose of 0.2 microg/pup per day (0.1 mg/kg) on alternate days from postnatal days 2 to 12. Fertility was tested at 80-240 days of age and tissues were examined at 96-255 days of age. DES caused malformation of the os penis, significant reductions in penile length, diameter, and weight, accumulation of fat cells in the corpora cavernosa penis, and significant reductions in weight of the bulbospongiosus and levator ani muscles in wild-type mice. Conversely, ERalphaKO mice treated with DES developed none of the above abnormalities. While nine out of ten male mice sired pups in the wild-type/control group, none did in the wild-type/DES group. ERalphaKO mice, despite normal penile development, are inherently infertile. Both plasma and intratesticular testosterone levels were unaltered in the DES-treated wild-type or DES-treated ERalphaKO mice when compared with controls, although testosterone concentration was much higher in the ERalphaKO mice. Hence, the resistance of ERalphaKO mice to developing penile abnormalities provides unequivocal evidence of an obligatory role for ERalpha in mediating the harmful effects of neonatal DES exposure in the developing penis.

Estrogen-induced abnormal accumulation of fat cells in the rat penis and associated loss of fertility depends upon estrogen exposure during critical period of penile development.

We previously reported that diethylstilbestrol (DES) or estradiol valerate (EV) exposure at a dose of 0.10-0.12 mg/kg, or higher, per day, on alternate days, from postnatal days 2-12, resulted in abnormal penis development and infertility (H. O. Goyal et al., 2005, J. Androl. 26, 32-43). The objective of this study was to identify a critical developmental period(s) during which EV exposure results in the observed penile abnormalities. Male pups received EV at a dose of 0.10-0.12 mg/kg on postnatal day(s) 1, 1-3, 4-6, 1-6, 7-12, 13-18, 19-24, or 25-30. Fertility was tested at 102-115 days of age and tissues were examined at 117-137 days. Both penile morphology and fertility were unaltered in rats treated with EV after 12 days of age. Conversely, except in rats treated on postnatal day 1 only, none of the males treated prior to 12 days of age sired pups, and all had abnormal penises, including varying degrees of abnormal accumulation of fat cells and loss of cavernous spaces and smooth muscle cells in the corpora cavernosa penis, which were maximal in the 1-6-day group. Also, the preputial sheath was partially released or its release was delayed, and the weight of the bulbospongiosus muscle was significantly reduced. Plasma testosterone (T) in the 1-6- and 4-6-day groups and intratesticular T in the 4-6-day group were significantly lower. The testosterone surge, characteristic of controls in the first week of life, was suppressed in the 1-3-day group. Estrogen receptor alpha mRNA expression was enhanced in the body of the penis in the 1-3-day group, but not in the 13-18-day group. Hence, EV exposure prior to 12 days of age (as short as 1-3 days postnatal), but not after 12 days of age, results in long-term abnormal penile morphology, characterized by abnormal accumulation of fat cells in the corpora cavernosa penis and, consequently, loss of fertility.

Exposure of neonatal male rats to estrogen induces abnormal morphology of the penis and loss of fertility.

The research objectives are to determine whether estrogen-induced infertility is associated with abnormal morphology of the penis and if morphological alterations can be reversed by testosterone (T). Male pups received diethylstilbestrol (DES) on alternate days from postnatal days 2 to 12. They received T or empty implants at 180 days, were tested for fertility at 188 days, and terminated at 200 days. While 5/7 control males sired pups, only 1/6 did in the DES group, and 0/8 in the DES plus T group. In addition to reductions in penile length and weight, the novel structural change induced by DES, and not reversed by T, was a replacement of cavernous spaces by fat cells in the penis body. Hence, T substitution for 8 days at adulthood did not reverse infertility in rats treated neonatally with DES and provided evidence that infertility probably resulted from absence of cavernous spaces and/or accumulation of fat cells in the penis body.

Abnormal morphology of the penis in male rats exposed neonatally to diethylstilbestrol is associated with altered profile of estrogen receptor-alpha protein, but not of androgen receptor protein: a developmental and immunocytochemical study.

Objectives of the study were to determine developmental changes in morphology and expression of androgen receptor (AR) and estrogen receptor (ER)alpha in the body of the rat penis exposed neonatally to diethylstilbestrol (DES). Male pups received DES at a dose of 10 microg per rat on alternate days from Postnatal Day 2 to Postnatal Day 12. Controls received olive oil vehicle only. Tissue samples were collected on Days 18 (prepuberty), 41 (puberty), and 120 (adult) of age. DES-induced abnormalities were evident at 18 days of age and included smaller, lighter, and thinner penis, loss of cavernous spaces and associated smooth muscle cells, and increased deposition of fat cells in the corpora cavernosa penis. Fat cells virtually filled the entire area of the corpora cavernosa at puberty and adulthood. Plasma testosterone (T) was reduced to an undetectable level, while LH was unaltered in all treated groups. AR-positive cells were ubiquitous and their profile (incidence and staining intensity) did not differ between control and treated rats of the respective age groups. Conversely, ERalpha-positive cells were limited to the stroma of corpus spongiosus in all age groups of both control and treated rats, but the expression in treated rats at 18 days was up-regulated in stromal cells of corpora cavernosa, coincident with the presence of morphological abnormalities. Hence, this study reports for the first time DES-induced developmental, morphological abnormalities in the body of the penis and suggests that these abnormalities may have resulted from decreased T and/or overexpression of ERalpha.

Neonatal estrogen exposure of male rats alters reproductive functions at adulthood.

The effects of neonatal exposure to different doses of diethylstilbestrol (DES) on the reproductive functions of male rats at adulthood were evaluated. Sprague-Dawley rats (5-8/group) received sc injections of 25 microl olive oil containing DES (Sigma Chemical Co., St. Louis, MO) at a dose of 10 microg, 1 microg, 100 ng, 10 ng, or 1 ng per rat on alternate days from Postnatal Days 2-12. Control animals received olive oil only. All animals were allowed to develop until 83-91 days of age; however, when they were 70 to 80 days old, four male rats each from the 10 microg, 1 microg, 100 ng, and control groups were cohabited with untreated 60- to 70-day-old females (1:1) for 12 days. At the end of cohabitation, both mated and unmated male rats were weighed, and blood and tissue samples were collected and processed. Results revealed that although sperm motility patterns and sperm morphology were adversely affected in the 10- microg group, other reproductive parameters, including 1). daily sperm production (DSP)/testis; 2). absolute and relative weights of the testis, epididymis, and seminal vesicle; and 3). sperm numbers in both regions of the epididymis declined significantly in a dose-dependent manner in the 10- and 1- microg groups. Conversely, in the <1- microg groups, none of these parameters (except DSP/testis and weight of the epididymis in the 100-ng group, and sperm numbers in the epididymis of the 100- and 10-ng groups) was different from controls. Generally, plasma testosterone levels decreased in the 10- and 1- microg groups, FSH level increased in the 10-microg group, and prolactin and LH levels were unaltered. In the fertility study, although each male in the 1-microg, 100-ng, and control groups produced a copulatory plug and impregnated a female, none could do so in the 10-microg group. The mean number of pups per litter was reduced to eight in the 1-microg group, in contrast to 15 each in the 100-ng and control groups. In conclusion, exposure of neonatal male rats to DES altered sperm motility patterns, sperm fertility (as evident from the reduced number of pups in the 1-microg group), and sexual behavior (as evident from the absence of copulatory plugs in the 10-microg group) and reduced weights of reproductive organs, DSP/testis, and sperm numbers in the epididymis. Whether these alterations/reductions persist in older rats (6-8 mo of age) is under investigation.

Expression and molecular characterization of estrogen receptor alpha messenger RNA in male reproductive organs of adult goats.

The fact that male estrogen receptor alpha (ERalpha) knockout mice are infertile indicates a role for this receptor in male reproduction. Here, objectives were to evaluate ERalpha expression in male goat reproductive tissues at the transcriptional level using RNase protection assay (RPA) and in situ hybridization (ISH), and to clone a partial cDNA for caprine ERalpha using reverse transcription-polymerase chain reaction (RT-PCR). For RPA and ISH procedures, a radiolabeled antisense cRNA probe, generated in vitro from the ovine oER8 cDNA template, was employed. Evaluations were made on individual samples obtained from adult goats. Labeled cRNA sense probe was used as a negative control in ISH. A 530-base pair amplicon was generated by RT-PCR from efferent ductules (EDs), epididymis (EP), and testis, cloned from the ED and EP, and sequenced. The caprine ERalpha (cERalpha) cDNA displayed 81%-96% sequence identity with that of other species. A signal indicative of ERalpha mRNA was identified by both RT-PCR and RPA in all tissues, but was strongest in the ED. Compared with ED, ERalpha signal was sixfold lower in the EP, and 66-fold lower in the testis. Similarly, strong ERalpha expression was observed in ED epithelium, whereas little or no signal was detected in EP or testis by ISH. Thus, among different segments of the male reproductive tract and testis, the highest level of ERalpha mRNA expression was found in epithelium of the ED.

Diethylstilbestrol-treated adult rats with altered epididymal sperm numbers and sperm motility parameters, but without alterations in sperm production and sperm morphology.

In this study, we characterized estrogenic effects of diethylstilbestrol (DES) on reproductive parameters in male rats to identify a minimal dose level that alters epididymal and sperm functions but has little or no effect on sperm production and/or spermatogenesis. Adult rats (five animals/group) received s.c. injections of 0.2 ml of corn oil containing DES at a rate of 1.0 mg, 200 microg, 40 microg, 8 microg, 1.6 microg, or 320 ng x rat(-1) x day(-1) for 12 days. The control group received corn oil only. DES effects were similar in the 8-microg group and higher dose groups and included significant (P < or = 0.05) reductions in 1) absolute and relative weights of the head and body of the epididymis (EP), tail of the EP, and seminal vesicle, 2) numbers of sperm in both regions of the EP, and 3) motility characteristics in sperm collected from the tail of the EP. Conversely, no significant changes were observed in relative testis weight, daily sperm production, spermatogenesis, seminiferous epithelial height in stage VII, and sperm morphology. All of the above parameters in the 1.6-microg group (except seminal vesicle weight) and 320-ng group were comparable to those of controls. Plasma testosterone (T) level was reduced to an almost undetectable level in the > or = 8-microg groups and to a very low level in the 1.6-microg group (0.35 vs. 2.36 ng/ml in controls or 320-ng group), but LH level was unaltered. In a parallel fertility study, males received DES at a rate of 40, 8, or 1.6 microg x rat(-1) x day(-1) for 12 days prior to and 12 days during cohabitation (1:1) with untreated females. Of the 15 females cohabited with treated males (5 females/dose), none in the 40-microg and 8-microg groups and 1 in the 1.6-microg group formed a copulatory plug and delivered 8 pups, in contrast to 5/5 copulatory plugs and 13-15 pups/litter in the controls. DES at a rate of 8 microg x rat(-1) x day(-1) for 12 days reduced EP weights, sperm numbers in the EP, and sperm motility patterns but caused minimal to no alterations in daily sperm production, spermatogenesis, or sperm morphology. Factors other than T, or in addition to lower T, may be responsible for DES-induced reproductive disorders (despite lower T, sperm contents and sperm motility patterns in the EP were normal in the 1.6-microg group). Deficits in EP sperm functions and/or sexual behavior (as evident from absence of copulatory plugs) probably accounted for reduced fertility in treated males.

Postnatal differentiation of efferent ductule epithelium in goats: a light microscopic and ultrastructural study.

Caprine efferent ductule epithelium contains ciliated and nonciliated cells. The latter cells are divided into three types: type II cells contain PAS-positive granules, type III cells contain PAS-negative vacuoles, and type I cells lack both granules and vacuoles (Goyal and Williams, Anat. Rec. 220:58-67). The objectives of this study are i) to determine when the epithelium differentiates into ciliated and nonciliated cells, ii) to determine when nonciliated cells acquire characteristics typical for type II and type III cells, and iii) to relate developmental changes in the epithelium with those in the testis. Testes and efferent ductules were examined at the light and electron microscopic levels in goats from 1-25 weeks of age. Efferent ductule epithelium contained ciliated and nonciliated cells as early as week 1. While ciliated cells were differentiated at week 1, differentiation of nonciliated cells did not occur until week > or =15. Differential features in ciliated cells included the presence of cilia at the apical border and an aggregation of mitochondria in the apical cytoplasm. Those in nonciliated cells included the presence of i) an endocytotic apparatus at week > or =15, ii) PAS-positive granules at week > or =15, and iii) PAS-negative vacuoles at week > or =25. The seminiferous tubules developed lumens at 12-15 weeks. Hence, while differentiation of ciliated cells occurred much before lumen formation in the seminiferous tubules, that of nonciliated cells coincided with, or occurred soon after, lumen formation, suggesting a role for testicular fluid contents in their differentiation. The goat efferent ductules can be characterized morphologically mature by 25 weeks.

Postnatal differentiation of the ductus deferens, tail of the epididymis, and distal body of the epididymis in goats occurs independently of rete testis fluid.

Observations from extratesticular rete-ligated, mature goats indicated that epithelial morphology in the tail of the epididymis can be maintained without any input from testicular fluid (Goyal et al., Acta Anat., 1994;150: 127-135). Hence, the objective of this study was to determine whether the tail of the epididymis and/or other regions of the male excurrent ducts can differentiate prior to the appearance of lumen in the seminiferous tubules, which is an indicator for the onset of seminiferous tubular fluid secretion. Based on age and scrotal circumference (SC), 20 male goats were divided into four groups of five animals each: 1-4 weeks (SC, 6.5-7.5 cm), 7-10 weeks (SC, 8.5-11.0 cm), 12-15 weeks (SC, 11.0-14.0 cm), and 15-25 weeks (SC, 16.0-19.0 cm). Tissues were collected from the testis, six regions of the epididymis (proximal, middle and distal head; proximal and distal body; and tail), and the ductus deferens, and were processed for light and electron microscopic examination. Changes in epithelial height and cytological features associated with absorption (microvilli, pinocytotic and coated vesicles) and protein secretion (RER, Golgi body) were used as markers for differentiation. Differentiation of all of these features was comparable to that observed in the 15-25-week-old animals in the ductus deferens by > or = 1 week, in the tail of the epididymis by > or = 7 weeks, in the distal body of the epididymis by > or = 12 weeks, and in the proximal body of the epididymis and all three regions of the head of the epididymis by > or = 15 weeks. Seminiferous tubules developed lumens between 12 and 15 weeks. In conclusion, epithelial differentiation in the ductus deferens, tail of the epididymis, and distal body of the epididymis follows a time-dependent, spatial, ascending order and is achieved before lumen formation in the seminiferous tubules. Conversely, epithelial differentiation in all three regions of the head and the proximal body of the epididymis occurs simultaneously and after lumen formation in the seminiferous tubules.

Regulation of androgen and estrogen receptors in male excurrent ducts of the goat: an immunohistochemical study.

BACKGROUND: Since androgens and/or estrogens must bind with specific receptors in order to elicit a response at the target organ(s), it is important to understand factors that regulate expression of androgen receptors (AR) and estrogen receptors (ER). Hence, the objective of the study is to determine the relative significance between circulating androgen (CA) and luminal androgen (LA) in maintaining normal expression of AR and ER in male excurrent ducts. METHODS: Mature Nubian goats were subjected for 15 days each to the following treatments: (1) bilateral orchidectomy, (2) bilateral orchidectomy and testosterone treatment, (3) unilateral ligation of the extratesticular rete, and (4) unilateral orchidectomy. Tissues from different segments of the excurrent ducts were fixed in 4% paraformaldehyde and embedded in Paraplast-plus. Antigenic sites for AR and ER were immunolocalized using PG-21 rabbit antirat/human antibody and H-222 rat antihuman monoclonal antibody, respectively. The avidin-biotin horseradish peroxidase procedure was used to identify positive immunoreactivity. Negative controls included incubation of sections with irrelevant IgG in place of primary antibody. RESULTS: In intact animals, whereas AR were found in epithelial, connective tissue, and peritubular smooth muscle cells of the efferent ductules, regions I-V of the epididymis, and ductus deferens, ER were confined to nonciliated cells of the efferent ductules. Bilateral orchidectomy caused a severe loss of both AR and ER staining. Testosterone replacement to orchidectomized animals restored staining of both AR and ER to the intact level. Neither unilateral ligation of the extratesticular rete nor unilateral orchidectomy had any effect on AR or ER immunostaining. CONCLUSION: Circulating androgen alone, without any input from luminal androgen or other rete fluid contents, can regulate expression of both androgen receptor and estrogen receptor.

Immunolocalization of androgen receptor and estrogen receptor in the developing testis and excurrent ducts of goats.

BACKGROUND: Because of the significance of androgens and estrogens in prenatal and postanatal differentiation of the testis and excurrent ducts, it is important to understand the developmental pattern of androgen receptor (AR) and estrogen receptor (ER) in these organs. METHODS: Tissues from 1-23-week-old goats were fixed in 4% paraformaldehyde and embedded in Paraplast-plus. Antigenic sites for AR and ER were immunolocalized using the PG-21 rabbit anti-rat/human antibody and the H-222 rat anti-human monoclonal antibody, respectively. The avidin-biotin horseradish peroxidase procedure was used to identify positive immunoreactivity. Controls included incubation of sections with irrelevant IgG in place of primary antibody. RESULTS: Within the testis, immunostaining for AR in the nuclei of Sertoli cells increased gradually from mild at week 1 to strong at week > or = 19. In contrast, nuclei of peritubular myoid cells and Leydig cells exhibited moderate to strong reaction for AR in all animals. Germ cells were negative. Within the rete testis, efferent ductules, regions I-V of the epididymis, and ductus deferens, nuclei of all epithelial cells, peritubular myoid cells, and intertubular connective tissue cells expressed moderate to strong staining for AR at all ages. ER were confined to nonciliated cells of the efferent ductules, which displayed moderate staining in all animals, beginning from week 1. CONCLUSIONS: Nuclear AR staining, found in all testicular cells (except germ cells) and excurrent duct cells examined, was observed to change in an age-related manner only in Sertoli cells, where staining intensity increased between week 1 and week 19. Staining for ER, confined to nonciliated epithelial cells of the efferent ductules, was not affected by postnatal age.

Immunolocalization of receptors for androgen and estrogen in male caprine reproductive tissues: unique distribution of estrogen receptors in efferent ductule epithelium.

Androgens and estrogens affect physiological processes in the testis and male excurrent duct system. This study was designed to identify and characterize distribution of androgen receptors (AR) and estrogen receptors (ER) in the reproductive organs of the male goat. Tissues, including testis, efferent ductules, epididymis (regions I-V), and ductus deferens, were obtained from five mature Nubian goats, fixed in 4% paraformaldehyde, and embedded in paraplast. Antigenic sites for AR were unmasked by microwave treatment (four times, 5 min each) of tissue sections immersed in 10 mM citrate (pH 6) and were detected using the PG-21 rabbit anti-rat/human antibody. Antigenic sites for ER were identified using the H-222 rat anti-human monoclonal antibody after tissue sections were treated with pronase (0.5 mg/ml, 37 degrees C, 8 min). Avidin-biotin horseradish peroxidase procedures were used to identify positive immunoreactivity. Irrelevant IgG was substituted for primary antibody in negative controls. Positive nuclear immunostaining for AR was observed in all types of epithelial cells, peritubular smooth muscle cells, and intertubular fibroblasts of the intratesticular rete, efferent ductules, epididymis (regions I-V), and ductus deferens, as well as in Sertoli, Leydig, and peritubular myoid cells and intertubular fibroblasts of the testis. In contrast, nuclear immunostaining for ER was confined to nonciliated cells of the efferent ductules. Thus, AR-positive cells are ubiquitously distributed in caprine testicular and excurrent ductular tissues, and ER-positive cells are unique to the efferent ductules. The caprine model should be useful in studies designed to determine mechanisms through which androgens and estrogens regulate development and function of the testes and excurrent ducts.