Reproductive Tract Development Research Articles

Development of a putative adverse outcome pathway network for male rat reproductive tract abnormalities with specific considerations for the androgen sensitive window of development

Structured approaches like the adverse outcome pathway (AOP) framework offer great potential for depicting complex toxicological processes in a manner that can facilitate informed integration of mechanistic information in regulatory decisions. While this concept provides a structure for organizing evidence and facilitates consistency in evidence integration; the process, inputs, and manner in which AOPs and AOP networks are developed is still evolving. Following the OECD guiding principles of AOP development, we propose three AOPs for male reproductive tract abnormalities and derive a putative AOP network. The AOPs were developed using a fundamental understanding of the developmental biology of the organs of interest, paying close attention to the gestational timing of key events (KEs) to very specifically inform the domain of life stage applicability for the key event relationships (KERs). Chemical stressor data primarily from studies on low molecular weight phthalates (LMWPs) served to ‘bound’ the pathways of focus in this dynamic period of development and were integrated with the developmental biology data through an iterative process to define KEs and conclude on the extent of evidence in support of the KERs. The AOPs developed describe the linkage between 1) a decrease in Insl3 gene expression and cryptorchidism, 2) the sustained expression of Coup-tfII and hypospadias and 3) the sustained expression of Coup-tfII and altered Wolffian duct development/ epididymal agenesis. A putative AOP network linking AOP2 and AOP3 through decreased steroidogenic biosynthetic protein expression and converging of all AOPS at the population level impaired fertility adverse outcome is proposed. The network depiction specifies and displays the KEs aligned with their occurrence in gestational time. The pathways and network described herein are intended to catalyze collaborative initiatives for expansion into a larger network to enable effective data collection and inform alternative approaches for identifying stressors impacting this sensitive period of male reproductive tract development.

Genital tract morphometry and haematology of male rabbits fed graded levels of cassava leaf meal

A study was conducted to investigate the effects of feeding graded levels of cassava leaf meal (CLM) on genital tract morphometry and haematology of twenty 8-week old male rabbits weighing between 815 to 910g. The rabbits were randomly assigned to four dietary treatments viz: T1 (control), T2, T3 and T4 with CLM inclusion levels of 0%, 9%, 18%, and 27%, respectively after balancing for live weight. Each rabbit was individually caged and served as a replicate in a completely randomized design. The feeding trial lasted for sixteen (16) weeks. Three (3) rabbits per treatment were slaughtered and evaluated for genital tract morphometry and haematological characteristics. The results showed that CLM had no significant effect (P>0.05) on testes, corpus, cauda, ductus and tunic weights. Positive relationships were observed between CLM in the diets and testes weight (r = 0.42; P<0.05) as well as cauda weights (r = 0.96; P<0.01). No significant effect (P>0.05) of diets were observed on packed cell volume, haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, platelets, white blood cells, neutrophils, lymphocytes, monocytes basophils and eosinophils. However negative relationships between levels of inclusion of CLM in diets and red blood cell, packed cell volume and haemoglobin were observed. It was concluded that the inclusion of up to 27% of CLM in bucks' diets is not detrimental to good health and normal reproductive tract development. It is recommended that further studies on the feeding potentials of cassava leaf meal at higher levels of inclusion and actual reproductive performance be investigated.

Estrogens and development of the rete testis, efferent ductules, epididymis and vas deferens

Estrogen has always been considered the female hormone and testosterone the male hormone. However, estrogen's presence in the testis and deleterious effects of estrogen treatment during development have been known for nearly 90 years, long before estrogen receptors (ESRs) were discovered. Eventually it was learned that testes actually synthesize high levels of estradiol (E2) and sequester high concentrations in the reproductive tract lumen, which seems contradictory to the overwhelming number of studies showing reproductive pathology following exogenous estrogen exposures. For too long, the developmental pathology of estrogen has dominated our thinking, even resulting in the “estrogen hypothesis” as related to the testicular dysgenesis syndrome. However, these early studies and the development of an Esr1 knockout mouse led to a deluge of research into estrogen's potential role in and disruption of development and function of the male reproductive system. What is new is that estrogen action in the male cannot be divorced from that of androgen. This paper presents what is known about components of the estrogen pathway, including its synthesis and target receptors, and the need to achieve a balance between androgen- and estrogen-action in male reproductive tract differentiation and adult functions. The review focuses on what is known regarding development of the male reproductive tract, from the rete testis to the vas deferens, and examines the expression of estrogen receptors and presence of aromatase in the male reproductive system, traces the evidence provided by estrogen-associated knockout and transgenic animal models and discusses the effects of fetal and postnatal exposures to estrogens. Hopefully, there will be enough here to stimulate discussions and new investigations of the androgen:estrogen balance that seems to be essential for development of the male reproductive tract.

Insight into a first trimester human placental mechanism of fetal endocrine disruption

Human chorionic gonadotropin (hCG) is a placental hormone produced during pregnancy which regulates in part the development of fetal reproductive tract. hCG is thought to mediate effects of prenatal phthalates on the fetus. We have shown previously that phthalate-hCG and hCG-neonatal genitalia associations are both sex-specific. The aim was to directly measure, within the relevant critical window in the first trimester, modification of hCG regulation by fetal sex and by phthalate levels. Placental tissue samples (N=96, 45% male) were donated by women undergoing elective pregnancy termination at 2 clinics between gestational weeks 7 - 14. Tissues were dissected, snap frozen and stored at -80 C. mRNAs were measured by qPCR: PPARG, CGA, CGB and 2 housekeeping genes, RN18S and UBP1. PPARG encodes a transcription factor that regulates expression of hCG (encoded by CGA and CGB). Quantitation was done by relative standard curve. Sex was determined by measuring 2 Y-linked genes by PCR. All data were normalized, transformed, and analyzed using linear regression. Placental phthalate levels in the same sample set are being analyzed currently. There was no difference in the means of mRNAs between male and female fetuses. We measured a strong interaction of PPARG and fetal sex in the correlation of PPARG and CGA/CGB (p-interaction<0.005). In male placentas, one log10 unit in PPARG was associated with a 2.53 (95% CI 1.80, 3.26) log10 increase in CGA and CGB levels. In female placentas, the same association was null (β= 0.37 log10 units, 95% CI -0.32, 1.05). Associations with placental phthalate levels are forthcoming. These data offer novel insight into the basis for commonly reported sex-specific associations between prenatal phthalates and child health outcomes. hCG offers promise as a molecular biomarker that can conceptually and quantitatively connect sex-specific effects of prenatal exposures on placental mechanisms, and on fetal outcomes.

Systematic comparison of the male reproductive tract in fetal and adult Wistar rats exposed to DBP and DINP in utero during the masculinisation programming window

This study investigates possible effects of in utero exposure of rats to a low dose (125 mg/kg bw/day) and a high dose (750 mg/kg bw/day) of Diisononyl phthalate (DINP) during the masculinisation programming window (MPW) which is embryonic days 15.5–18.5 (e15.5 - e18.5). Dibutyl phthalate (DBP) was used at a high dose level (750 mg/kg bw/day) as an established positive control substance for anti-androgenic effects on the developing male reproductive tract.We focussed on the MPW and measured a multitude of biological endpoints at various life stages and applied state of the art histopathology staining techniques to refine the characterization of potential changes to the testis, beyond what is currently available with DINP. If DINP can mediate testicular dysgenesis (TDS) disorders, this exposure window would be sufficient to induce androgen impacts and alter male reproductive tract development as shown earlier in this validated experimental model with DBP.Overall, the results of this systematic comparison provide convincing evidence on the differences between the effects occurring with DBP and DINP. In contrast to what was seen with DBP, DINP did not cause cryptorchidism or hypospadias, had no effect on anogenital distance/anogenital index (AGD/AGi) and Leydig cell aggregates on e17.5 and e21.5 did not increase. With DINP no reduction of intratesticular testosterone, no effects on sperm motility and sperm count and no effect on adult testosterone or luteinizing hormone (LH) levels were seen. Our results demonstrate that DINP does not cause the adverse reproductive effects known to occur with DBP, a well-established endocrine disruptor.

Transforming growth factor beta signaling and decidual integrity in mice†.

Transforming growth factor beta (TGFβ) signaling regulates multifaceted reproductive processes. It has been shown that the type 1 receptor of TGFβ (TGFBR1) is indispensable for female reproductive tract development, implantation, placental development, and fertility. However, the role of TGFβ signaling in decidual development and function remains poorly defined. Our objective is to determine the impact of uterine-specific deletion of Tgfbr1 on decidual integrity, with a focus on the cellular and molecular properties of the decidua during development. Our results show that the developmental dynamics of the decidua is altered in TGFBR1 conditionally depleted uteri from embryonic day (E) 5.5 to E8.5, substantiated by downregulation of genes associated with inflammatory responses and uterine natural killer cell abundance, reduced presence of nondecidualized fibroblasts in the antimesometrial region, and altered decidual cell development. Notably, conditional ablation of TGFBR1 results in the formation of decidua containing more abundant alpha smooth muscle actin (ACTA2)-positive cells at the peripheral region of the antimesometrial side versus controls at E6.5-E8.5. This finding is corroborated by upregulation of a subset of smooth muscle marker genes in Tgfbr1 conditionally deleted decidua at E6.5 and E8.5. Moreover, increased cell proliferation and enhanced decidual ERK1/2 signaling were found in Tgfbr1 conditional knockout mice upon decidual regression. In summary, conditional ablation of TGFBR1 in the uterus profoundly impacts the cellular and molecular properties of the decidua. Our results suggest that TGFBR1 in uterine epithelial and stromal compartments is important for the integrity of the decidua, a transient but crucial structure that supports embryo development.

Antral follicular count has limited impact in the selection of more fertile beef heifers

The selection of replacements using applications such as reproductive tract scores and antral follicle count may be useful to eliminate less fertile animals before breeding. Previously, we reported that heifers with increased numbers of follicles achieved puberty earlier than heifers with diminished numbers of follicles. We, therefore, hypothesized that heifers that achieved a reproductive tract score ≥4 earlier in the breeding season would have greater numbers of follicles than heifers that achieved a reproductive tract score ≥4 later in the breeding season. The uterus and ovaries of three hundred and fifty beef heifers from a commercial farm were evaluated by transrectal ultrasonography 20 days before breeding and parameters of growth and development were recorded. The antral follicle count (AFC) of both ovaries was recorded and the heifers assigned a reproductive tract score (1 to 5). Only heifers with a reproductive tract score ≥4 entered to two fixed timed artificial insemination (FTAI) programs. The first group (FTAI1,n = 269) started the day of the beginning of the breeding period and the second group (FTAI2, n = 61) one month later. The rest were naturally mated (no FTAI, n = 20). Serial pregnancy scanning was performed throughout the breeding season and 45 days after bull removal to estimate the day of conception. Data indicative of growth and development were analysed using the REG and GLM procedures of SAS with FTAI (0, 1 and 2), AFC (low, medium and high), and the interaction as the independent variables. Reproductive tract development was negatively associated to AFC (P>0.0001). There was no interaction between FTAI and AFC (P>0.11). Heifers inseminated at FTAI1 were heavier and taller, had greater deposition of fat and muscle, and conceived earlier than those inseminated at FTAI2 (P<0.05); however, there was no difference in AFC between heifers inseminated in FTAI1 and FTAI2 (15.6 ± 0.4 vs. 16.7 ± 0.9 follicles, respectively; P = 0.3). Furthermore, conception rates did not differ statistically between heifers in FTAI1 and FTAI2 (P = 0.12). Antral follicle count influenced growth and carcass traits (P>0.05) and heifers in the High AFC class conceived approximately 1 week earlier than heifers in the Low and Medium AFC class (P<0.05). We conclude that pubertal heifers have less AFC, thus similar numbers were observed for heifers in both FTAI programs and no differences in pregnancy rate. Antral follicle count may allow for the selection of heifers that conceive earlier, but may result in a lower pregnancy rate.

YAP and TAZ are required for the postnatal development and the maintenance of the structural integrity of the oviduct.

The development of the Müllerian ducts into the female reproductive tract requires the coordination of multiple signaling pathways that regulate proliferation, apoptosis and differentiation. The Hippo pathway has been reported to interact with several pathways with established roles in Müllerian duct development; yet, its potential roles in reproductive tract development and function remain mostly uncharacterized. The objective of this study was therefore to characterize the roles of the Hippo transcriptional coactivators YAP and TAZ in the female reproductive tract using transgenic mouse models. This report shows that the concomitant conditional inactivation of Yap and Taz in the mouse Müllerian duct mesenchyme results in postnatal developmental defects of the oviduct. Most notably, discontinuities in the myosalpinx layer lead to the progressive formation of cystic dilations of the isthmus. These defects prevented embryo transport and subsequent implantation in older animals, causing infertility. The loss of YAP/TAZ did not appear to affect other biological processes known to be required for the maintenance of oviductal wall integrity, such as TGF-β/SMAD and Notch signaling and the biogenesis of miRNA, suggesting that the Hippo pathway acts independently of these processes to direct oviduct development. Taken together, these results suggest redundant and essential roles for YAP and TAZ in the postnatal development of the oviduct and the maintenance of its structural integrity.

Gestational vinclozolin exposure suppresses fetal testis development in rats

Vinclozolin is a common dicarboximide fungicide used to protect crops from diseases. It is also an endocrine disruptor and is thought to be related to abnormalities of the reproductive tract. However, its mechanism of inducing abnormalities of the male reproductive tract is still unclear. The purpose of this study was to study the effect of gestational vinclozolin exposure on the development of rat fetal Leydig cells. Female pregnant Sprague-Dawley rats were exposed to vinclozolin (0, 25, 50, and 100 mg/kg body weight/day) by gavage from gestational day 14–21. Vinclozolin dose-dependently reduced serum testosterone levels at doses of 50 and 100 mg/kg and the anogenital distance at 100 mg/kg. RNA-seq, qPCR, and Western blotting showed that vinclozolin down-regulated the expression of Nr5a1, Sox9, Lhcgr, Cyp11a1, Hsd3b1, Hsd17b3, Amh, Pdgfa, and Dhh and their encoded proteins. Vinclozolin reduced the number of NR2F2-positive stem Leydig cells at a dose of 100 mg/kg and enhanced autophagy in the testes. In conclusion, vinclozolin disrupts reproductive tract development and testis development in male fetal rats via several pathways.

SIX1 cooperates with RUNX1 and SMAD4 in cell fate commitment of M\xfcllerian duct epithelium

During female mammal reproductive tract development, epithelial cells of the lower Müllerian duct are committed to become stratified squamous epithelium of the vagina and ectocervix, when the expression of ΔNp63 transcription factor is induced by mesenchymal cells. The absence of ΔNp63 expression leads to adenosis, the putative precursor of vaginal adenocarcinoma. Our previous studies with genetically engineered mouse models have established that fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK), bone morphogenetic protein (BMP)/SMAD, and activin A/runt-related transcription factor 1 (RUNX1) signaling pathways are independently required for ΔNp63 expression in Müllerian duct epithelium (MDE). Here, we report that sine oculis homeobox homolog 1 (SIX1) plays a critical role in the activation of ΔNp63 locus in MDE as a downstream transcription factor of mesenchymal signals. In the developing mouse reproductive tract, SIX1 expression was restricted to MDE within the future cervix and vagina. SIX1 expression was totally absent in SMAD4 null MDE and was reduced in RUNX1 null and FGFR2 null MDE, indicating that SIX1 is under the control of vaginal mesenchymal factors: BMP4, activin A and FGF7/10. Furthermore, Six1, Runx1, and Smad4 gene-dose-dependently activated ΔNp63 expression in MDE within the vaginal fornix. Using a mouse model of diethylstilbestrol (DES)-associated vaginal adenosis, we found DES action through epithelial estrogen receptor α (ESR1) inhibits activation of ΔNp63 locus in MDE by transcriptionally repressing SIX1 and RUNX1 in the vaginal fornix.

Uterovaginal Anomalies: A Guide for the Generalist Obstetrician-Gynecologist.

Congenital gynecologic anomalies result from interruption of embryologic development of the female reproductive tract. The anomalies may be hymenal, vaginal, cervical, or uterine. The impact of these anomalies is variable: some are asymptomatic, incidental findings that require no intervention, others require simple surgical management, while some complex anomalies may require a multidisciplinary approach with extensive surgical expertise for optimal outcomes. Uterovaginal anomalies may occur in isolation or in association with other malformations, such as renal anomalies. The origin, presentation, evaluation and treatment of these conditions are reviewed here.

Phenotypic Characterization and Comparison of Cystic Fibrosis Rat Models Generated Using CRISPR/Cas9 Gene Editing

Animal models of cystic fibrosis (CF) are essential for investigating disease mechanisms and trialing potential therapeutics. This study generated two CF rat models using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 gene editing. One rat model carries the common human Phe508del (ΔF508) CF transmembrane conductance regulator (CFTR) mutation, whereas the second is a CFTR knockout model. Phenotype was characterized using a range of functional and histologic assessments, including nasal potential difference to measure electrophysiological function in the upper airways, RNAscope in situ hybridization and quantitative PCR to assess CFTR mRNA expression in the lungs, immunohistochemistry to localize CFTR protein in the airways, and histopathologic assessments in a range of tissues. Both rat models revealed a range of CF manifestations, including reduced survival, intestinal obstruction, bioelectric defects in the nasal epithelium, histopathologic changes in the trachea, large intestine, and pancreas, and abnormalities in the development of the male reproductive tract. The CF rat models presented herein will prove useful for longitudinal assessments of pathophysiology and therapeutics.

Endometriosis, endocrine disrupters, and epigenetics: an investigation into the complex interplay in women with polybrominated biphenyl exposure and endometriosis.

Endocrine disrupting compounds (EDCs) have been shown to affect multiple biologic processes especially steroid-hormone processes. We sought to determine differences in DNA methylation exists between women with and without endometriosis following exposure to polybrominated biphenyl (PBB). Cross-sectional study of 305 females in the Michigan PBB Registry. DNA was extracted, and DNA methylation was interrogated using the MethylationEPIC BeadChip (Illumina, San Diego, California). Demographic data was analyzed using Chi-squared and T tests. Linear regressions were performed for each cytosine-guanine dinucleotide (CpG) site, modeling the logit transformation of the β value as a linear function of the presence of endometriosis. Sensitivity analyses were conducted controlling for estradiol levels and menopausal status. Replication study performed evaluating for any association between CpGs reported in the literature and our findings. In total, 39,877 CpGs nominally associated with endometriosis (p < 0.05) after adjusting for age and cellular heterogeneity, although none remained significant after correction for multiple comparisons (FDR < 0.05). Pathway analysis of these CpGs showed enrichment in 68 biologic pathways involved in various endocrine, immunologic, oncologic, and cell regulation processes as well as embryologic reproductive tract development and function (FoxO, Wnt, and Hedgehog signaling). We identified 42,261 CpG sites in the literature reported to be associated with endometriosis; 2012 of these CpG sites were also significant in our cohort. We found 39,877 CpG sites that nominally associated with endometriosis (p < 0.05) after adjusting for age and cellular heterogeneity; however, none remained significant after correction for multiple comparisons (FDR < 0.05).

Effects of reproductive tract development and hormonal treatment on estrous synchronization in heifers

Evaluation of the reproductive tract development (RTD) is a criterion rarely used when heifers are included in estrous synchronization programs. The objective of the present study was to determine the effect of RTD (mature and immature) and hormonal treatment (EC and EB) on estrous expression rate and pregnancy rate in Bos indicus heifers under tropical conditions. RTD was evaluated with an ultrasound and classified as mature (n=99) or immature (n=101). Heifers received an intravaginal device (DIB®; day 0), 2 mg of estradiol benzoate (EB) and 250 µg of cloprostenol (PGF2α). The DIB was removed on day 7 post insertion, and 250 µg of PGF2α was applied and heifers were divided into two groups: One group of heifers received 0.5 mg of estradiol cypionate (EC group), and on day 8, a second group of heifers received 1 mg of estradiol benzoate (EB group). Estrus was detected by visual observation, and all heifers were inseminated at a fixed-time (FTAI). Fifteen days after insemination, bulls were introduced to the treatment groups. Data were analyzed using general modeling and binary logistic regression procedures. The percentage of estrus was similar for both mature and immature heifers (P&gt;0.05), however, the heifers treated with EB had 17% animals in estrus, than the heifers that received EC (P &lt;0.05). The pregnancy rate after FTAI (74.8%) and total pregnancy (FTAI + natural mating = 91.9%) was greater for the group of heifers with a mature reproductive tract (P&lt;0.05). The hormone treatment had not effect (P&gt;0.05) on the pregnancy rate. In conclusion, the heifers with mature reproductive tracts had the highest pregnancy rate after artificial insemination and total pregnancy. Hormonal treatment did not influence the pregnancy rate.

A novel long non-coding RNA, Leat1, causes reduced anogenital distance and fertility in female mice

Defective anorectal and urogenital malformations are some of the most severe congenital anomalies encountered in children. Only a few molecular cues have been identified in early formation of the female urogenital system. Here we describe a novel long non-coding RNA molecule known as Leat1 (long non-coding RNA, EphrinB2 associated transcript 1). This lncRNA is syntenic with EfnB2 (which encodes EphrinB2) and expressed during embryonic development of the genital tubercle. While lncRNAs have varied functions, many are known to regulate their neighbouring genes. Eph/Ephrin bidirectional signaling molecules mediate many patterning pathways in early embryonic development, including cloacal septation and urethral development. Here we investigate the role of Leat1 and its possible regulation of EphrinB2 during development of the female reproductive tract. We show that a loss of Leat1 leads to reduced EfnB2 expression in the developing female genital tubercle, reduced anogenital distance and decreased fertility.

EFECTO DEL DESARROLLO DEL TRACTO REPRODUCTIVO Y TRATAMIENTO HORMONAL SOBRE LA ACTIVIDAD OVÁRICA DE NOVILLAS BOS INDICUS

&lt;p&gt;Evaluation of the reproductive tract development (RTD) is a criterion rarely used when heifers are included in estrous synchronization programs. The objective was to determine the effect of RTD and hormonal treatment (HT) on estrous and ovarian activity in &lt;em&gt;Bos indicus&lt;/em&gt; heifers under humid tropical conditions. Twenty-four &lt;em&gt;Bos indicus&lt;/em&gt; heifer were synchronized using a bovine intravaginal device (BID), divided according to RTD (immature or mature) and HT (estradiol cypionate or estradiol benzoate). Estrous was recorded from 24 to 60 h after BID removal. Follicular monitoring was evaluated by ultrasound every 12 h from BID removal until ovulation. Blood samples were taken on day 0, 3, 8 and 15 post BID removal to estimate progesterone (P&lt;sub&gt;4&lt;/sub&gt;) concentration. Data were analyzed by analysis of variance and logistic regression procedures. There was no interaction between factors (P &amp;gt; 0.05). The highest percentage of estrous was found in heifers with mature RTD (91.7%, P &amp;lt; 0.05). The interval BID withdrawal to estrous (50.0±2.6 h), the proportion of heifers that ovulated (100%), the interval BID withdrawal-ovulation (75.0±2.1 h), and the time between onset of estrous and ovulation (24.5±2.2 h) were similar between immature and mature reproductive tracts and between HT. In addition, the growth of dominant follicles and P&lt;sub&gt;4&lt;/sub&gt; concentration were similar for both study group. In conclusion, RTD had effect on estrous behavior, however, ovulation, diameter of dominant follicle and plasma P&lt;sub&gt;4&lt;/sub&gt; concentration of both groups (RTD and HT) were similar on estrous synchronization, in &lt;em&gt;Bos indicus&lt;/em&gt; heifers under humid tropical conditions.&lt;/p&gt;

A Hormone That Lost Its Receptor: Anti-Müllerian Hormone (AMH) in Zebrafish Gonad Development and Sex Determination.

Fetal mammalian testes secrete Anti-Müllerian hormone (Amh), which inhibits female reproductive tract (Müllerian duct) development. Amh also derives from mature mammalian ovarian follicles, which marks oocyte reserve and characterizes polycystic ovarian syndrome. Zebrafish (Danio rerio) lacks Müllerian ducts and the Amh receptor gene amhr2 but, curiously, retains amh To discover the roles of Amh in the absence of Müllerian ducts and the ancestral receptor gene, we made amh null alleles in zebrafish. Results showed that normal amh prevents female-biased sex ratios. Adult male amh mutants had enormous testes, half of which contained immature oocytes, demonstrating that Amh regulates male germ cell accumulation and inhibits oocyte development or survival. Mutant males formed sperm ducts and some produced a few offspring. Young female mutants laid a few fertile eggs, so they also had functional sex ducts. Older amh mutants accumulated nonvitellogenic follicles in exceedingly large but sterile ovaries, showing that Amh helps control ovarian follicle maturation and proliferation. RNA-sequencing data partitioned juveniles at 21 days postfertilization (dpf) into two groups that each contained mutant and wild-type fish. Group21-1 upregulated ovary genes compared to Group21-2, which were likely developing as males. By 35 dpf, transcriptomes distinguished males from females and, within each sex, mutants from wild types. In adult mutants, ovaries greatly underexpressed granulosa and theca genes, and testes underexpressed Leydig cell genes. These results show that ancestral Amh functions included development of the gonadal soma in ovaries and testes and regulation of gamete proliferation and maturation. A major gap in our understanding is the identity of the gene encoding a zebrafish Amh receptor; we show here that the loss of amhr2 is associated with the breakpoint of a chromosome rearrangement shared among cyprinid fishes.

Carryover effects of pre- and postweaning planes of nutrition on reproductive tract development and estrous cycle characteristics in Holstein heifers

The objectives of this study were to investigate the carryover effects of differing pre- and postweaning planes of nutrition on prepubertal reproductive tract development and postpubertal estrous cycle characteristics in Holstein heifers. Heifer calves (n = 36) were randomly assigned to receive either a low or a high (5 or 10 L of whole milk/d, respectively) preweaning diet from 1 to 7 wk of age and either a low or a high (70 or 85% of concentrate dry total mixed ration, respectively) postweaning diet from 11 to 25 wk of age. Starting at wk 26 of age, heifers were subjected to weekly transrectal ultrasonography until wk 33 or until first ovulation to assess endometrial thickness and ovarian follicular count and size in the prepubertal phase. In a subset of heifers (n = 28), ovarian ultrasonography continued weekly until at least the second ovulation was confirmed; thereafter, ovarian dynamics (through ultrasonography) and blood progesterone (P4) were assessed every 2 d throughout 1 complete estrous cycle in the postpubertal phase. In the prepubertal phase, endometrial thickness (12.0 ± 0.4 vs. 10.8 ± 0.3 mm) and largest follicle size (11.8 ± 0.3 vs. 10.9 ± 0.2 mm) were greater in heifers fed the high postweaning diet than in those fed the low postweaning diet. Furthermore, the number of class 2 (6-9 mm) follicles was greater in heifers fed the high preweaning diet than in those fed the low preweaning diet (1.6 ± 0.1 vs. 1.1 ± 0.1), whereas the number of class 3 (>9 mm) follicles was greater in heifers fed the high postweaning diet than in those fed the low postweaning diet (1.2 ± 0.1 vs. 1.0 ± 0.1). In the postpubertal phase, overall corpus luteum and P4 dynamics did not differ among pre- or postweaning treatments; however, P4 at 4 d preceding luteolysis was lesser in heifers fed the high postweaning diet than in those fed the low postweaning diet (6.1 ± 0.4 vs. 7.7 ± 0.4 ng/mL). In addition, compared with heifers fed the low postweaning diet, those fed the high postweaning diet had a greater number of antral follicles (31.4 ± 2.2 vs. 21.4 ± 2.3) and tended to have more class 3 follicles (3.6 ± 0.3 vs. 2.7 ± 0.3). Results indicate positive carryover effects of increasing the preweaning plane of nutrition from 5 to 10 L of whole milk/d on prepubertal follicular growth in Holstein heifers. Furthermore, an increased postweaning plane of nutrition (85 vs. 70% of concentrate dry total mixed ration) advanced reproductive development through greater endometrial thickness and follicular growth in the prepubertal phase and increased the population of antral follicles in the postpubertal estrous cycle.

Effects of phthalates on bovine primary testicular culture and spermatozoa.

Among environmental endocrine-active chemicals, phthalates, commonly known as plasticizers, disrupt the development of the male reproductive tract. In this study, the effects of phthalates (DIBP, BBP, DINP, DBP, DEP, DEHP and DMP) were evaluated on cultures of bovine primary male reproductive cells (n = 3) and spermatozoa (n = 4). Epididymal (caput and corpus epididymis), testicular (parenchymal and mediastinal/tubular) and vas deferens cells (VDC) were prepared from samples collected from slaughterhouse. Second part of caput epididymis which have fewer amount of principal cells, were found to be less affected compared to the first part except DEHP; while corpus epididymis was found to be more affected with IC50 values below 0.976ng/mL (except for DEP at 4.97ng/mL). In testicular parenchymal cells, IC50 ranged from 0.15 to 4.11ng/mL and for mediastinum from 0.01 to 7.31ng/mL; where cytotoxic effects were more evident in mediastinal section. Least cytotoxic and even proliferational effects (DEHP, DMP and DEP) were observed in VDC, the muscular tube carrying sperm from epididymis to the ejaculatory duct. Least spermiotoxic phthalate was DBP (3.928ng/mL); while DINP (0.550ng/mL) induced highest cytotoxic effect on bovine spermatozoa. Differences in the cellular structure and/or the androgen receptor distribution effect the toxicity of phthalates. Our preliminary findings on bovine spermatozoa indicate possible morphological and motility alterations; which challenges further investigation of the transition of phthalates on semen straws used in cryopreservation. Increase of exposure to environmental contaminants raise the issue of the requirement of a new perspective on reproductive health, species and tissue specific differences should further be emphasized.

Carlo Acerini - raging against the dying of the light.

Carlo Acerini - raging against the dying of the light.