Critical Period Of Sexual Differentiation Research Articles

Self-fertilization in a gonochoristic fish yellow catfish

Sex control techniques are in great demand for aquaculture because of the huge benefits of mono-sex production. In a previous study on yellow catfish (Tachysurus fulvidraco), intersex individuals were produced by administrating 17α-methyltestosterone (MT) or aromatase inhibitor letrozole (LZ). The ovarian and testicular portions of intersexual gonads developed well and were completely separated, arousing our interest in producing self-fertilizable fish and clone-like fish populations. In this study, yellow catfish fry were treated with a combination of MT and LZ at a low dose (5 mg kg−1 MT, 300 mg kg−1 LZ) at a low water temperature (27.0 °C, mixed sex-producing temperature, LL group) and a high temperature (33.5 °C, male-producing temperature, HL group), and with a high dose (10 mg kg−1 MT, 600 mg kg−1 LZ) at a low water temperature (LH group), during 12–65 days post-hatching, the critical period of sex differentiation. Both XX and XY intersex were produced with different percentages at the same time through co-treatment with MT and LZ, with or without the help of male-producing temperature. The highest percentage of XY intersex (53% in XY, 36% in XX and XY) and XX intersex (47% in XX, 25% in XX and XY) was produced in LH and HL groups, respectively. Results of gonadal morphology, histology, and sperm dynamic analysis indicate that some XX and XY intersex individuals were capable of self-fertilization through artificial insemination. The reproductive performance of these intersex individuals was then investigated by injecting spawning agents, dissecting gonads, and artificially fertilizing eggs with their own sperm. The XX, XY, and YY genotypic individuals were produced by the self-fertilization of XY intersex individuals. The present study successfully produced high percentages of XX and XY intersex and conducted self-fertilization by mating the eggs and sperm of a single intersex individual. Self-fertilization in fish will show great potential in aquaculture genetics and breeding, for example, generating highly homozygous (or ‘‘clonal’’) genetic material, producing mono-sex populations, and identifying unknown sex determination modes. In particular, the cloning method of producing genetic materials will be greatly beneficial for maintaining advantageous traits through self-fertilization generation after generation.

Sex Reversal Induced by Dietary Supplementation with 17α-Methyltestosterone during the Critical Period of Sex Differentiation in Oriental River Prawn (Macrobrachium nipponense).

The steroid 17α-methyltestosterone (MT) inhibits ovarian function and is often used to induce sex reversal artificially in vertebrates. In the present study, different concentrations of MT were added as dietary supplementation, and the effects on sex ratio, growth, and gonadal development were examined. After 40 days, the sex ratio (male:female) in each group increased at different degrees with 50 (1.36:1), 100 (1.57:1), and 200 (2.61:1) mg/kg MT, and neo-males with testis-ovary coexistence were observed in the 200 mg/kg MT group. Furthermore, 50 and 100 mg/kg MT could induce female reversion in neo-males. Histologically, the development of the testes in experimental groups was slower, but the ovaries of the experimental and control groups had similar developmental rates. The expression levels of DMRT11E, Foxl2, and SoxE1 in males at 200 mg/kg MT were 8.65-, 3.75-, and 3.45-fold greater than those of the control group. In crustaceans, sex reversal through vertebrate sex hormones can be observed. Neo-males (sex-reversed female prawns) were maintained by exogenous androgen, and over-reliance led to slow testis growth, small body size, and low growth rate, but sperm was still produced. In female prawns, MT inhibited ovary development and promoted growth.

DNA methylation and demethylation shape sexual differentiation of neurochemical phenotype

Some of the best-studied neural sex differences depend on differential cell death in males and females, but other sex differences persist even if cell death is prevented. These include sex differences in neurochemical phenotype (i.e., stable patterns of gene expression). Work in our laboratory over the last several years has tested the hypothesis that sex differences in DNA methylation early in life underlie sexual differentiation of neuronal phenotype. We have shown that 1) expression of enzymes that place or remove DNA methylation marks is greatest during the first week of life in the mouse brain and overlaps with the perinatal critical period of sexual differentiation; 2) a transient inhibition of DNA methylation during neonatal life abolishes several sex differences in cell phenotype in the mouse hypothalamus; 3) both DNA methylation and de-methylation contribute to the development of neural sex differences; and 4) the effects of DNA methylation and de-methylation are brain region- and cell type-specific.

High-temperature stress will put the thermo-sensitive teleost yellow catfish (Tachysurus fulvidraco) in danger through reducing reproductivity

Recently, concerns for species that sex differentiation is influenced by temperature in the context of global warming have increased because disrupted operational sex ratios could threaten population maintenance. In contrast, little attention has been given to the reproductive ability of populations that experienced elevated temperatures. In this study, we demonstrated that high temperature (HT) would decrease population size via three different aspects of reproductive ability for the first time. We show that, in a thermo-sensitive teleost yellow catfish, a short period of HT (+3 °C) exposure during the critical period of sex differentiation leads to a different percentage of masculinization of XX genotypic females (1–23%) in wet-lab and natural water bodies. Combining the results of gonadal appearance, histology, sperm parameters, and fertilization rate, we found that XX pseudo-males induced by HT display significantly discounted fertility and reproductive performance compared to XY normal males. We demonstrate that the survival of the XY genotype is lower than XX genotype under environmental stress, including HT, hypoxia, and parasite infection, and the differential survival seems unrelated to male-biased sexual size dimorphism. The mathematical model predicts that the phenotypic female percent will be stabilized at 50% and the population will be sustainably maintained when masculinizing force is less than 0.5, while HT will put the population in danger when the masculinizing force exceeds 0.5. However, when we combine the real-world data of reproductive ability and mathematic model, our results suggest the population size decreases and the long-term survival of the studied species are threatened under the projected pace of increasing temperature. These findings will be useful for understanding the long-term effects of increasing temperature on sex ratio, reproduction and population maintenance in teleost.

Особливості розвитку репродуктивної системи щурів нащадків чоловічої статі, що народжені від батька, який до спаровування отримував фітоестрогени

The purpose of the study was the research of the influence of rats father's excessive consumption of phytoestrogens on somato-sexual development and reproductive function of male offspring. Materials and methods. Male parents were fed with a mixture of phytoestrogens before mating with intact females at a dose of 20 mg/kg of body weight for 30 days. In reproductive animals, reproductive function was studied for such characteristics as sexual behavior, fertility, spermogram, the mass of androgen-dependent organs was determined, and the level of sex hormones was determined. Experimental parents were divided into two groups: 1) intact, 2) males who received excess phytoestrogens. After the birth of male offspring from these parents, their reproductive system was examined. Results and discussion. Prolonged administration of phytoestrogens to mature male parents (parents) causes absolute and relative hyperestrogenism (five-fold increase in E2 and vice versa reduction of T/E2 three-fold), which leads to a decrease in copulatory activity (reduction of the number of nurseries and intromissions by 28%), violation of spermatogenesis (reduction of sperm concentration by 39%, doubling the proportion of abnormal forms), at the same time does not affect the reproductive potential of male who were born to fathers who received an excess consumption of phytoestrogens. The presence of excess compounds with estrogenic activity in the diet of adult males in the preconsumption period leads to the birth of male offspring, which have reduced testosterone levels (25%) in the critical period of sexual differentiation of the brain (5 days of life). Descendants of the male sex of the phytoestrogenized father have a normal course of somatic development but are characterized by impaired sexual development (less anogenital distance by 13%) and delayed lowering of the testicles into the scrotum, lower body weight in adulthood (28.2% in 150 day rats). Reproductive function of the offspring of phytoestrogenized father is characterized by differences in sexual behavior: less copulatory activity – 50%, less number intromission, the number of males with ejaculation is 10%; delayed sexual reactions; and lower reproductive potential (two-fold reduction of the integrated indicator of potential fertility of animals). Decreased reproductive potential of the offspring, who were born to fathers who received an excess consumption of phytoestrogens, occurs due to a decrease in the proportion of effective fertilization (pregnant females among those fertilized only 60%), indicating negative changes in sperm, the development of which, starting from the stage of spermatocytes, occurred in conditions of absolute and relative hyperestrogenism. Conclusion. The male who were born to fathers who received an excess consumption of phytoestrogens, have complications in the functioning of the reproductive system

Gonadal development and molecular analysis revealed the critical window for sex differentiation, and E2 reversibility of XY-male spotted scat, Scatophagus argus

In vertebrate the development of gonad into testis or ovary requires the combined effect of genes and hormones to trigger differentiation from a bipotential gonadal primordium. Knowledge of the gonad development process is vital in studies on fish sex determination and differentiation. The spotted scat (Scatophagus argus) is an economically important marine fish with an XX/XY sex-determining system and exhibits sexual growth dimorphism in favor of females. Meanwhile, information on gonad development and gene expression patterns in the early gonads is unknown. This study explores the pattern of spotted scat early gonad development by morphological and molecular means. In the undifferentiated gonads, the number of germ cells in females is not significantly different from males. At age 3 mph (months post-hatch), the number of germ cells in females was significantly higher than in males. The sex differentiation began in females before males with the proliferation of primary oocyte and spermatocytes at 3.5 and 4 mph, respectively. In the early gonads, dmrt1 and amh were significantly higher in XY-fish, while cyp19a1a and foxl2 were higher in the XX-fish. Within the critical period of sex differentiation, estradiol treatment was able to sex-reverse XY-male to XY-female. In the sex-reversed group, male (dmrt1 and amh) and female (cyp19a1a and foxl2) related genes were suppressed and increased, respectively. 17α-Methyltestosterone or aromatase inhibitor (letrozole) could not induce sex reversal in XX-fish due to the absence of the male-specific dmrt1 required for testes development. Conclusively, this study identifies the critical window for sex differentiation and significantly higher germ cell numbers in future females than males. We provide evidence that the phenotypic XY-male is reversible, while the phenotypic XX-female is irreversible. Dmrt1 is necessary for testis differentiation, amh in males, and cyp19a1a and foxl2 in females could also be used as molecular markers in early gonads. This study explicitly presents valuable information for research development on sex determination and differentiation and sex control in spotted scat.

Exposure of zebrafish to elevated temperature induces sex ratio shifts and alterations in the testicular epigenome of unexposed offspring

Accumulating evidence shows that environmental changes can affect population sex ratios through epigenetic regulation of gene expression in species where sex depends on both genetic and environmental cues. Sometimes, altered sex ratios persist in the next generation even when the environmental cue is no longer present (a multigenerational effect). However, evidence of transgenerational effects (i.e., beyond the first non-exposed generation), which tend to be paternally transmitted, is scarce and a matter of debate. Here, we used the AB strain of zebrafish, where sex depends on both genetic and environmental influences, to study possible multi- (to the F1) and transgenerational (to the F2) effects of elevated temperature during the critical period of sex differentiation. From eight initial different families, five were selected in order to capture sufficient variation between the sex ratio of the control group (28 °C) and the group exposed to elevated (35 °C) temperature only at the parental (P) generation. Results showed a consistent increase in the proportion of males in the P generation in all five families as a result of heat treatment. Sex ratios were then determined in the F1 and F2 offspring derived from both above groups, which were all raised at 28 °C. A persisting male-skewed sex ratio in the 35°C-derived, unexposed offspring of the F1 generation was observed in three families, denoting family-dependent multigenerational effects. However, no transgenerational effects were observed in the F2 generation of any family. DNA methylation was also assessed in the testis of P, F1 and F2 males derived from exposed and non-exposed fathers and grandfathers. DNA methylation was significantly decreased only in the testis of the 35°C-derived males in the F1 generation but not of the F2 generation and, surprisingly, neither in the 35°C-exposed males of the P generation. Taken together, our results show great interfamily variation, not only in sex ratio response to elevated temperature, but also on its multigenerational effects, denoting a strong influence of genetics. Alterations in the testicular epigenome in F1 males calls for attention to possible, previously unnoticed, effects of temperature in the unexposed offspring of heat-exposed parents in a global warming scenario.

Neuroendocrine-Immune Crosstalk Shapes Sex-Specific Brain Development.

Sex is an essential biological variable that significantly impacts multiple aspects of neural functioning in both the healthy and diseased brain. Sex differences in brain structure and function are organized early in development during the critical period of sexual differentiation. While decades of research establish gonadal hormones as the primary modulators of this process, new research has revealed a critical, and perhaps underappreciated, role of the neuroimmune system in sex-specific brain development. The immune and endocrine systems are tightly intertwined and share processes and effector molecules that influence the nervous system. Thus, a natural question is whether endocrine-immune crosstalk contributes to sexual differentiation of the brain. In this mini-review, we first provide a conceptual framework by classifying the major categories of neural sex differences and review the concept of sexual differentiation of the brain, a process occurring early in development and largely controlled by steroid hormones. Next, we describe developmental sex differences in the neuroimmune system, which may represent targets or mediators of the sexual differentiation process. We then discuss the overwhelming evidence in support of crosstalk between the neuroendocrine and immune systems and highlight recent examples that shape sex differences in the brain. Finally, we review how early life events can perturb sex-specific neurodevelopment via aberrant immune activation.

Role for Kisspeptin and Neurokinin B in Regulation of Luteinizing Hormone and Testosterone Secretion in the Fetal Sheep.

Evidence suggests that the hypothalamic-pituitary-gonadal (HPG) axis is active during the critical period for sexual differentiation of the ovine sexually dimorphic nucleus, which occurs between gestational day (GD) 60 and 90. Two possible neuropeptides that could activate the fetal HPG axis are kisspeptin and neurokinin B (NKB). We used GD85 fetal lambs to determine whether intravenous administration of kisspeptin-10 (KP-10) or senktide (NKB agonist) could elicit luteinizing hormone (LH) release. Immunohistochemistry and fluorescent in situ hybridization (FISH) were employed to localize these peptides in brains of GD60 and GD85 lamb fetuses. In anesthetized fetuses, KP-10 elicited robust release of LH that was accompanied by a delayed rise in serum testosterone in males. Pretreatment with the GnRH receptor antagonist (acyline) abolished the LH response to KP-10, confirming a hypothalamic site of action. In unanesthetized fetuses, senktide, as well as KP-10, elicited LH release. The senktide response of females was greater than that of males, indicating a difference in NKB sensitivity between sexes. Gonadotropin-releasing hormone also induced a greater LH discharge in females than in males, indicating that testosterone negative feedback is mediated through pituitary gonadotrophs. Kisspeptin and NKB immunoreactive cells in the arcuate nucleus were more abundant in females than in males. Greater than 85% of arcuate kisspeptin cells costained for NKB. FISH revealed that the majority of these were kisspeptin/NKB/dynorphin (KNDy) neurons. These results support the hypothesis that kisspeptin-GnRH signaling regulates the reproductive axis of the ovine fetus during the prenatal critical period acting to maintain a stable androgen milieu necessary for brain masculinization.

Discovery and Profiling of microRNAs at the Critical Period of Sex Differentiation in Xanthoceras sorbifolium Bunge

Research Highlights: The critical period of sex differentiation in Xanthoceras sorbifolium was investigated. Multiple microRNAs (miRNAs) were identified to influence female and male flower development, with some complementary functions. Background and Objectives: Xanthoceras sorbifolium Bunge is widely cultivated owing to its multipurpose usefulness. However, as a monoecious plant, the low female–male flowers ratio and consequent low seed yield are the main bottlenecks for industrial-scale development of seed utilization. MiRNAs play crucial regulatory roles in flower development and sex differentiation; therefore, we evaluated the roles of miRNAs in the critical period of sex differentiation in X. sorbifolium. Materials and Methods: Four small RNA libraries for female and male flower buds of the critical period of sex differentiation were constructed from paraffin-embedded sections. The miRNAs were characterized by high-throughput sequencing, and differentially expressed miRNAs were validated by reverse transcription-quantitative polymerase chain reaction. Results: There were obvious differences in male and female pistil and stamen flower buds, with elongated inflorescence and clear separation of flower buds marking the critical period of sex differentiation. A total of 1619 conserved miRNAs (belonging to 34 families) and 219 novel miRNAs were identified. Among these, 162 conserved and 14 novel miRNAs exhibited significant differential expression in the four libraries, and 1677 putative target genes of 112 differentially expressed miRNAs were predicted. These target genes were involved in diverse developmental and metabolic processes, including 17 miRNAs directly associated with flower and gametophyte development, mainly associated with carbohydrate metabolism and glycan biosynthesis and metabolism pathways. Some miRNA functions were confirmed, and others were found to be complemented. Conclusions: Multiple miRNAs closely related to sex differentiation in X. sorbifolium were identified. The theoretical framework presented herein might guide sex ratio regulation to enhance seed yield.

Male-biased zebrafish sex differentiation and metabolomics profile changes caused by dydrogesterone

Some progestins, including the widely used dydrogesterone (DDG), have been shown to cause male-biased sex ratio in teleost. However, there is a gap to fully understand the mechanisms of the sex differentiation disturbance by progestins, particularly from the metabolic aspect. We thus aimed to examine the sex changes by exposing zebrafish embryos to 4.4 (L), 44 (M) and 440 (H) ng/L DDG for up to 140 days, and investigated metabolomic profile changes during the critical period of sex differentiation at fry stage (35 dpf). DDG increased the percentage of male zebrafish in a dose-dependent manner, with 98% male fish in the high concentration group. In zebrafish fry, DDG increased the levels of some free fatty acids, monoglycerides, acylcarnitines, organic acids, free amino acids, while decreased lysophospholipids, uric acid and bile acids. DDG exposure also decreased the nucleoside monophosphates and UDP-sugars while increased nucleosides and their bases. These metabolite changes, namely increase in n-3 PUFAs (polyunsaturated fatty acids), myo-inositol, taurine, palmitoleic acid, oleic acid, lactic acid, fumaric acid, and uracil, and decrease in uric acid and bile acids, might account for the male-biased sex ratio in zebrafish. It appears that many of these metabolites could inhibit several pathways that regulate zebrafish gonad differentiation, including NF-κB/COX-2 and Wnt/β-catenin pathways, and activate p53 pathway. Thus we proposed a hypothesis that DDG might induce oocytes apoptosis through the above pathways and finally lead to female-to-male sex reversal. The results from this study suggest that DDG at environmentally relevant concentrations could affect zebrafish metabolomic profiles and finally disturb fish sex differentiation.

Mast Cells in the Developing Brain Determine Adult Sexual Behavior.

Many sex differences in brain and behavior are programmed during development by gonadal hormones, but the cellular mechanisms are incompletely understood. We found that immune-system-derived mast cells are a primary target for the masculinizing hormone estradiol and that mast cells are in turn primary mediators of brain sexual differentiation. Newborn male rats had greater numbers and more activated mast cells in the preoptic area (POA), a brain region essential for male copulatory behavior, than female littermates during the critical period for sexual differentiation. Inhibiting mast cells with a stabilizing agent blunted the masculinization of both POA neuronal and microglial morphology and adult sex behavior, whereas activating mast cells in females, even though fewer in number, induced masculinization. Treatment of newborn females with a masculinizing dose of estradiol increased mast cell number and induced mast cells to release histamine, which then stimulated microglia to release prostaglandins and thereby induced male-typical synaptic patterning. These findings identify a novel non-neuronal origin of brain sex differences and resulting motivated behaviors.SIGNIFICANCE STATEMENT We found that immune-system-derived mast cells are a primary target for the masculinizing hormone estradiol and that mast cells are in turn primary mediators of brain sexual differentiation. These findings identify a novel non-neuronal origin of brain sex differences and resulting motivated behaviors.

Programming of the reproductive axis by hormonal and genetic manipulation in mice

In mammals, the reproductive function is controlled by the hypothalamic–pituitary–gonadal axis. During development, mechanisms mediated by gonadal steroids exert an imprinting at the hypothalamic–pituitary level, by establishing sexual differences in the circuits that control male and female reproduction. In rodents, the testicular production of androgens increases drastically during the fetal/neonatal stage. This process is essential for the masculinization of the reproductive tract, genitals and brain. The conversion of androgens to estrogens in the brain is crucial for the male sexual differentiation and behavior. Conversely, feminization of the brain occurs in the absence of high levels of gonadal steroids during the perinatal period in females. Potential genetic contribution to the differentiation of brain cells through direct effects of genes located on sex chromosomes is also relevant. In this review, we will focus on the phenotypic alterations that occur on the hypothalamic–pituitary–gonadal axis of transgenic mice with persistently elevated expression of the human chorionic gonadotropin hormone (hCG). Excess of endogenously synthesized gonadal steroids due to a constant hCG stimulation is able to disrupt the developmental programming of the hypothalamic–pituitary axis in both transgenic males and females. Locally produced estrogens by the hypothalamic aromatase might play a key role in the phenotype of these mice. The ‘four core genotypes’ mouse model demonstrated a potential influence of sex chromosome genes in brain masculinization before critical periods of sex differentiation. Thus, hormonal and genetic factors interact to regulate the local production of the neurosteroids necessary for the programming of the male and female reproductive function.

A single neonatal administration of soybean oil and/or tamoxifen affects permanently the testis histomorphology in adult rats.

The aim of this study was to determine the effect of tamoxifen (Tx) and its vehicle, soybean oil (SO), during the critical period of hypothalamic sexual differentiation in newborn male rats, regarding gonadal histomorphology during adulthood. The animals were randomly divided into 3 groups (n = 5 each). An hour after birth, one group was treated subcutaneously with 200 μg of Tx, using commercial SO (20 uL) as a vehicle; another group was treated with only 20 μL of SO; the control group received no treatment. All rats were weighed and sacrificed by cervical dislocation on day 90 post-treatment. Testicles were removed, weighed and processed for histological evaluation. The single administration of Tx and/or SO during the critical period of sexual differentiation of the hypothalamus permanently altered testicular histomorphology, spermatogenesis, and body weight in adulthood. Alterations included vacuolization and reduction in the number of spermatogonia and Sertoli cells. The administration of Tx reduced the testicular weight, the diameter and area of the seminiferous tubules, and the height of the germinal epithelium, and increased the intertubular space. Soybean oil by itself reduced the number of spermatocytes and spermatids more than Tx did. There was no effect on the number of Leydig cells. The possibility that soybean oil can act as an endocrine disruptor deserves greater attention and opens the possibility for the development of new methods of pest control.

A single neonatal administration of soybean oil and/or tamoxifen affects permanently the testis histomorphology in adult rats.

Veterinaria México OA ISSN: 2448-6760Cite this as:González González A, González Padilla E, Fierro Fierro F, Juárez Mosqueda M de L, Pérez Rivero JJ, Vergara Onofre M. A single neonatal administration of soybean oil and/or tamoxifen affects permanently the testis histomorphology in adult rats. Veterinaria México OA. 2016;3(2). doi: 10.21753/vmoa.3.2.364The aim of this study was to determine the effect of tamoxifen (Tx) and its vehicle, soybean oil (SO), during the critical period of hypothalamic sexual differentiation in newborn male rats, regarding gonadal histomorphology during adulthood. The animals were randomly divided into 3 groups (n = 5 each). An hour after birth, one group was treated subcutaneously with 200 μg of Tx, using commercial SO (20 uL) as a vehicle; another group was treated with only 20 μL of SO; the control group received no treatment. All rats were weighed and sacrificed by cervical dislocation on day 90 post-treatment. Testicles were removed, weighed and processed for histological evaluation. The single administration of Tx and/or SO during the critical period of sexual differentiation of the hypothalamus permanently altered testicular histomorphology, spermatogenesis, and body weight in adulthood. Alterations included vacuolization and reduction in the number of spermatogonia and Sertoli cells. The administration of Tx reduced the testicular weight, the diameter and area of the seminiferous tubules, and the height of the germinal epithelium, and increased the intertubular space. Soybean oil by itself reduced the number of spermatocytes and spermatids more than Tx did. There was no effect on the number of Leydig cells. The possibility that soybean oil can act as an endocrine disruptor deserves greater attention and opens the possibility for the development of new methods of pest control.Figure 1. Representative hematoxylin-eosin-stained histological sections of testicles collected from the control group (A y B), rats treated with SO (C y D) and rats treated with Tx (E y F). A, C and E, 200X. B, D and F, 400X. T: Tubule. LC: Leydig cells. Histological changes induced by Tx and/or SO treatments can be observed. Black arrows point to the largest intertubular space. White arrows in E and F indicate the ungrouping of the LC. Asterisks indicate the vacuoles in the seminiferous tubules.

Juvenile exposure to vinclozolin shifts sex ratios and impairs reproductive capacity of zebrafish.

Exposure to endocrine disruptors during critical periods of development can impact the sustainability of wild fish populations. Anti-androgenic compounds have received less attention, but are capable of modulating gonad differentiation and maturation, and impairing reproduction in fish. The fungicide vinclozolin (VZ) has been shown to impair reproduction in adult fish, but less is known about its effects following exposure earlier in development. Here we show that waterborne exposure to 400μg VZ/L during critical periods of sex differentiation (21-35 days post fertilization) permanently shifts sex ratios towards females, and alters the maturation of the gonad. Both fecundity and fertility were reduced, even when oogenesis and spermatogenesis recover and sperm motility is not altered. These results demonstrate the need to better understand the impacts of early exposure to anti-androgenic compounds on fish.

Dose-dependent acceleration in the delayed effects of neonatal oral exposure to low-dose 17α-ethynylestradiol on reproductive functions in female Sprague-Dawley rats.

Xenoestrogen exposure during the critical period of sexual differentiation of the brain causes delayed effects on female reproduction. We investigated the internal dose of orally administered ethynylestradiol (EE) during the critical period and its delayed effects by administering 0 (vehicle control), 0.4, or 2 μg/kg EE to female Sprague-Dawley rats for 5 days from postnatal day (PND) 1. Determination of serum EE level 24 hr after the initial dosing and 6 and 24 hr after the final dosing of 2 μg/kg indicated that the administered EE entered the circulation and cleared after every administration. Although the treatment did not affect physical development, including growth, eyelid opening, and vaginal opening, the estrous cycle was arrested from postnatal week (PNW) 12 even with 0.4 μg/kg EE, with an inverse correlation between doses and arresting ages. Although ovarian morphology at PNW 22-23 indicated that the treatment caused long-term anovulation and cystic follicle formation, the number of primordial follicles at PNW 22-23 was similar among the groups. Because this number was lower than that at PND 10 in all groups, primordial follicles may have been consumed under long-term anovulation. The treatment also caused other abnormalities, including mammary gland hyperplasia, increase in pituitary and liver weights, and decrease in the uterine weight. Because the highest circulating EE level in the 2 μg/kg-treated neonates is considered to be comparable to the physiological range of estradiol-17β, we concluded that a slight increase in the circulating estrogens during the neonatal period exerts irreversible delayed effects.

Molecular cloning and expression analysis of 17β-hydroxysteroid dehydrogenase 1 and 12 during gonadal development, recrudescence and after in vivo hCG induction in catfish, Clarias batrachus

In teleosts, the levels of steroids during critical period of sex differentiation are critical for gonadogenesis. Hence, steroidogenesis and expression of steroidogenic enzyme genes are very critical for gonadal development and function. In this regard, 17β-HSDs are important as they are involved in both 17β-estradiol (E2) and testosterone (T) biosynthesis. Full length cDNAs of 17β-HSD 1 (1791bp) and 12 (1073bp) were cloned from catfish gonads which encodes a protein of 295 and 317 amino acids, respectively. To understand the importance of these enzymes in teleost reproduction, mRNA expression was analyzed during gonadal development, seasonal reproductive cycle and after human chorionic gonadotropin (hCG) induction. Phylogenetic analysis revealed that the 17β-HSD 1 and 12 share high homology with their respective 17β-HSD forms from other teleosts and both the 17β-HSD forms belong to short chain dehydrogenase/reductase family. Tissue distribution analysis showed that the 17β-HSD 1 expression was higher in ovary and gills, while 17β-HSD 12 was higher expressed in testis, ovary, brain, intestine and head kidney compared to other tissues analyzed. Developing and mature ovary showed higher expression of 17β-HSD 1, while 17β-HSD 12 was higher in testis than the ovary of corresponding stages. Further, 17β-HSD 1 and 12 transcripts together with E2 and T levels were found to be modulated during different phases of the seasonal reproductive cycle. Expression of 17β-HSD 1 and 12 was upregulated after hCG induction which shows possible regulation by gonadotropin. Our findings suggest that 17β-HSD 1 and 12 might play important role in regulating gonadal development and gametogenesis through modulation of sex steroid levels.

Simvastatin and dipentyl phthalate lower ex vivo testicular testosterone production and exhibit additive effects on testicular testosterone and gene expression via distinct mechanistic pathways in the fetal rat.

Sex differentiation of the male reproductive tract in mammals is driven, in part, by fetal androgen production. In utero, some phthalate esters (PEs) alter fetal Leydig cell differentiation, reducing the expression of several genes associated with steroid synthesis/transport, and consequently, lowering fetal androgen and Insl3 hormone levels. Simvastatin (SMV) is a cholesterol-lowering drug that directly inhibits HMG-CoA reductase. SMV may also disrupt steroid biosynthesis, but through a different mode of action (MOA) than the PEs. As cholesterol is a precursor of steroid hormone biosynthesis, we hypothesized that in utero exposure to SMV during the critical period of sex differentiation would lower fetal testicular testosterone (T) production without affecting genes involved in cholesterol and androgen synthesis and transport. Secondly, we hypothesized that a mixture of SMV and a PE, which may have different MOAs, would reduce testosterone levels in an additive manner. Pregnant Sprague Dawley rats were dosed orally with SMV, dipentyl phthalate (DPeP), or SMV plus DPeP from gestational days 14-18, and fetuses were evaluated on GD18. On GD18, SMV lowered fetal T production and serum triglycerides, low density lipoprotein, high density lipoprotein, and total cholesterol levels, and downregulated two genes in the fetal testis that were different from those altered by PEs. When SMV and DPeP were administered as a mixture, fetal T production was significantly reduced in an additive manner, thus demonstrating that a mixture of chemicals can induce additive effects on fetal T production even though they display different MOAs.

Histological characterization of gonadal sex differentiation inNibea albiflora

The aim of this study was to investigate the period of sex differentiation and the pattern of gonadal differentiation in Nibea albiflora using histological techniques. The fish were raised in temperature between 19.5 and 21.5°C. The promordial gonads formed at the posterior region of the abdomen and the fish remained undifferentiated till the 36 day post hatching (dph) with a total length (TL) of 21.20 ± 3.90 mm. Ovarian differentiation precedes testicular differentiation, as usual in teleost fish. Oocytes undergoing meiosis at zygotene (bouquet) and pachytene stages were firstly observed when the total length reached 33.05 ± 4.61 mm at 40 dph, and the formation of the ovarian cavity began simultaneously at 46 dph with total length of 49.40 ± 4.83 mm. When the mean total length reached 78.05 ± 5.24 mm (61 dph), ovarian cavity grew bigger, and oocytes were firstly detected. By contrast, signs of morphological differentiation in the presumptive testis are not observed until their total length reached 69.18 ± 5.26 mm at 55 dph. The unrestricted lobular organization of the testis is evident at the stage of TL = 78.05 ± 5.24 mm (61 dph). These findings indicate that N. albiflora is a differentiated gonochorist and the critical period of sex differentiation in N. albiflora occurs at the stage of TL = 21.20 ± 3.90 mm (36 dph).