Gene mutations, including different alleles of the same gene, are tremendously useful in deconstructing complex developmental systems, such as reproduction, into component molecular pathways. For this reason, great effort has been devoted in the past three decades to biased (reverse genetic) and unbiased (forward genetic) searches for new genes that impactmammalian reproduction and fertility. These efforts have more recently been complemented with international efforts to systematically mutate all mouse genes and to determine their phenotypes (essentially a hybrid of forward and reverse genetics). Here, we survey the available data on the relative productivity of these approaches in identifying fertility genes, estimate the number of protein-coding genes essential for fertility of males and females, and predict the next major directions in the genetics of reproduction and fertility.

Summary Sentence

Strategies to identify the entire repertoire of mammalian genes required for fertility are reviewed.

Selection of a dominant follicle from a wave of follicles is manifested by diameter deviation between future dominant (F1) and largest subordinate (F2) follicles. On day –1 or 0 (day 0 = beginning of deviation), growth rate of F1 continues and growth rate of F2 decreases. Deviation occurs during the decline in the wave-stimulating FSH surge when F1 reaches means of 8.5, 10.5, and 22.5 mm in heifers, women, and mares, respectively. Diameter of F1 at the FSH peak vs at deviation is proportionally similar among these monovular species. In conventional deviation, F1 usually emerges first. In F1,F2-switched deviation, F2 is usually first to emerge and to reach a diameter characteristic of deviation. On day –1 or 0, the larger F2 and the smaller F1 switch so that the formerly larger F2 becomes subordinate and the formerly smaller F1 becomes dominant. In heifers and mares, the profile and prominence of the FSH surge are similar between deviation classes. Surge location relative to deviation differs so that the surge ends earlier in switched deviation. When the larger F2 reaches a diameter characteristic of deviation, FSH concentration is too low for continued growth of F2. The decrease in FSH ceases (heifers) or increases (mares) presumably from a decrease in FSH inhibitors; therefore, F1 continues to grow and becomes dominant. The frequency of switched deviation (e.g., 16 to 37% among species) can be problematic in follicle-selection research. Switching is a natural model for study of the interplay between follicles and FSH.

Cysteine-X-cysteine (CXC) motif chemokine ligand 12 (CXCL12) and its receptor, CXC chemokine receptor type 4 (CXCR4), are involved in regulating the proliferation, migration, and survival of trophoblast cells and the maternal immune response in humans and mice. The present study examined the expression, regulation, and function of CXCL12 and CXCR4 at the maternal–conceptus interface during pregnancy in pigs. The endometrium expressed CXCL12 and CXCR4 mRNAs with the greatest CXCL12 abundance on Day 15 of pregnancy. CXCL12 protein was localized mainly in endometrial epithelial cells, while CXCR4 protein was localized in subepithelial stromal cells, vascular endothelial cells, and immune cells in blood vessels in the endometrium during the estrous cycle and pregnancy. CXCL12 protein was detected in uterine flushing on Day 15 of pregnancy. The conceptus during early pregnancy and chorioallantoic tissues during mid-to-late pregnancy expressed CXCL12 and CXCR4. Interferon-γ increased the abundance of CXCL12, but not CXCR4 mRNA in endometrial explants. Recombinant CXCL12 (rCXCL12) protein dose-dependently increased migration of cultured porcine trophectoderm cells and peripheral blood mononuclear cells (PBMCs). Furthermore, rCXCL12 caused migration of T cells, but not natural killer cells, in PBMCs. This study revealed that interferon-γ -induced CXCL12 and its receptor, CXCR4, were expressed at the maternal–conceptus interface and increased the migration of trophectoderm cells and T cells at the time of implantation in pigs. These results suggest that CXCL12 may be critical for the establishment of pregnancy by regulating trophoblast migration and T cell recruitment into the endometrium during the implantation period in pigs.

Summary Sentence

A chemokine CXCL12 induced by interferon-γ of conceptus origin in the endometrium is involved in activation of trophectoderm cell migration and recruitment of T cells at the maternal–conceptus interface in pigs.

Reported classifications of germinal vesicle (GV) chromatin configurations in pig oocytes were not done by uniform standards and they were not well correlated with oocyte competence. In this study, GV chromatin of pig oocytes was classified into nonsurrounded nucleolus (NSN), surrounded nucleolus (SN), partly NSN (pNSN) and SN (pSN), prematurely condensed NSN (cNSN), pNSN (cpNSN) and pSN (cpSN), and early diakinesis (ED) patterns. During in vitro maturation in 199 medium, NSN oocytes from 1 to 2 mm follicles went consecutively through pNSN, pSN, cpSN, and ED before undergoing GV breakdown, and chromatin in some SN oocytes from 3 to 6 mm follicles re-decondensed into a re-decondensation (RDC) configuration. Under unfavorable conditions such as follicle atresia, ovary handling or maturation in simple MEM medium, however, premature chromatin condensation occurred, forming cNSN, cpNSN, and cpSN patterns. While all NSN and pNSN and some pSN and RDC oocytes actively transcribed, no cNSN, cpNSN, or cpSN oocytes showed transcription. Maturation and embryo culture suggested that SN and pSN oocytes were more competent than NSN and pNSN oocytes; cpSN oocytes were more competent than cNSN/cpNSN oocytes; and only RDC oocytes could develop into blastocysts. It is concluded that the newly classified chromatin configurations are more closely correlated with oocyte competence than those reported previously.

Summary Sentence

The newly classified GV chromatin configurations in pig oocytes are more closely correlated with oocyte transcriptional activity and developmental competence than previously reported ones.

Mechanisms regulating sperm motility activation are generally known in oviparous fishes, but are poorly understood in viviparous species. The mechanism of osmotic-shock induced signaling for oviparous fishes is not suitable for viviparous fishes which activate sperm motility within an isotonic environment. In addition, the presence of sperm bundles in viviparous fishes further complicates study of sperm activation mechanisms. The goal of this study was to establish methodologies to detect intracellular Ca²⁺ signals from sperm cells within bundles, and to investigate the signaling mechanism of sperm activation of viviparous fish using Redtail Splitfin (Xenotoca eiseni) as a model. Motility was assessed by classification of bundle dissociation and computer-assisted sperm analysis, and intracellular Ca²⁺ was assessed using the fluorescent probe Fura-2 AM. Bundle dissociation and sperm motility increased with extracellular Ca²⁺ and pH levels. Intracellular Ca²⁺ signals were detected from sperm within bundles, and increased significantly with extracellular Ca²⁺ and pH levels. Major channel blockers known to inhibit Ca²⁺ influx (NiCl₂, ruthenium red, GdCl₃, SKF-96365, nimodipine, verapamil, methoxyverapamil, mibefradil, NNC 55–0396, ω-Conotoxin MVIIC, bepridil, and 2-APB) failed to inhibit Ca²⁺ influx, except for CdCl₂, which partially inhibited the influx. We propose a novel mechanism for motility regulation of fish sperm: an alkaline environment in the female reproductive tract opens Ca²⁺ channels in the sperm plasma membrane without osmotic shock, and the Ca²⁺ influx functions as a second messenger to activate motor proteins controlling flagella movement.

Summary Sentence

Extracellular alkalinization can induce Ca²⁺ influx, which activates sperm within bundles of a viviparous fish.

The sperm-borne oocyte-activating factor (SOAF) resides in the sperm perinuclear theca (PT). A consensus has been reached that SOAF most likely resides in the postacrosomal sheath (PAS), which is the first region of the PT to solubilize upon sperm–oocyte fusion. There are two SOAF candidates under consideration: PLCZ1 and WBP2NL. A mouse gene germline ablation of the latter showed that mice remain fertile with no observable phenotype despite the fact that a competitive inhibitor of WBP2NL, derived from its PPXY motif, blocks oocyte activation when coinjected with WBP2NL or spermatozoa. This suggested that the ortholog of WBP2NL,WBP2, containing the same domain and motifs associated with WBP2NL function, might compensate for its deficiency in oocyte activation. Our objectives were to examine whether WBP2 meets the developmental criteria established for SOAF and whether it has oocyte-activating potential. Immunoblotting detected WBP2 in mice testis and sperm and immunofluorescence localized WBP2 to the PAS and perforatorium of the PT. Immunohistochemistry of the testes revealed that WBP2 reactivity was highest in round spermatids and immunofluorescence detected WBP2 in the cytoplasmic lobe of elongating spermatids and colocalized it with the microtubular manchette during PT assembly. Microinjection of the recombinant forms of WBP2 and WBP2NL into metaphase II mouse oocytes resulted in comparable rates of oocyte activation. This study shows that WBP2 shares a similar testicular developmental pattern and location with WBP2NL and a shared ability to activate the oocyte, supporting its consideration as a mouse SOAF component that can compensate for a WBP2NL.

Summary Sentence

WBP2 and WBP2NL share sperm location, development origins, and oocyte-activating ability in mouse.

Exposure to estrogenic endocrine disrupting chemicals (EDCs) during in utero development has been linked to the increasing incidence of disorders of sexual development. Hypospadias, the ectopic placement of the urethra on the ventral aspect of the penis, is one of the most common DSDs affecting men, and can also affect women by resulting in the misplacement of the urethra. This study aimed to comprehensively assess the resulting hypospadias phenotypes in male and female mice exposed in utero from embryonic day 9.5 to 19.5 to the potent estrogenic endocrine disruptor, diethylstilbestrol, at a high, clinically relevant dose, and a low, previously untested dose, administered via water. The anogenital distance of male pups was significantly reduced and hypospadias was observed in males at a high frequency. Females exhibited hypospadias and urethral-vaginal fistula. These results demonstrate the ability of an estrogen receptor agonist to disrupt sexual development in both male and female mice, even at a low dose, administered via drinking water.

Summary Sentence

In utero exposure to clinically relevant and low levels of diethylstilbestrol disrupts genital tubercle development in both male and female mice.

The objectives of the study were to compare the presence and localization of ovulation-inducing factor (OIF)/nerve growth factor (NGF) inmale reproductive organs and determine the abundance in ejaculates of species representative of both spontaneous and induced ovulators. We hypothesized that the protein is a widely conserved component of semen among mammals, but is most abundant in camelids. Immunohistochemical analysis was performed on tissues from the male reproductive system of llamas, rats, cattle, bison, elk, and white-tailed deer (n = 2 males/species), and the abundance of OIF/NGF in the seminal plasma of camelids (llamas and alpacas), cattle, horses, and pigs (n = 69, 53, 24, and 16 ejaculates, respectively) were quantified by radioimmunoassay. Based on immunoreactivity in both the glandular epithelium and glandular lumen, the prostate gland was the main source of seminal OIF/NGF in llamas, the vesicular gland and ampullae in bovids (cattle and bison), and the ampullae and prostate in cervids (elk and white-tailed deer). Camelid and bovine seminal plasma induced dendritic growth in the PC₁₂ differentiation bioassay, but no effect was observed with equine or porcine seminal plasma. The concentration of OIF/NGF was 10 times higher in camelid than bovine seminal plasma (1.2 ± 0.21 vs. 0.10 ± 0.03; P < 0.05); OIF/NGF was not detected in equine or porcine ejaculates by radioimmunoassay. Based on tissue localization, abundance, and bioactivity, we conclude that OIF/NGF is a common protein within the male accessory glands among species, and its abundance in camelids, bovids, and cervids suggests an important role in the mechanisms of ovulation in both induced and spontaneous ovulators.

Summary Sentence

Ovulation-inducing factor/nerve growth factor is conserved in the reproductive tissue of mammalian species.

A variety of mechanisms are involved in sex determination in vertebrates. The orange-spotted grouper (Epinephelus coioides), a teleost fish, functions first as females and later as a male and is an ideal model to investigate the regulation of sexual fate. Here, we report female-to-male sex reversal in juvenile orange-spotted groupers caused by overexpressing anti-Müllerian hormone (Amh). Tissue distribution analyses showed that amh and amhrII primarily expressed in the gonad, and expression level in the testis wasmuch higher than that in the ovary. In gonads, the expression of amh was located in the Sertoli cells around spermatogonia of the testis and in the zona pellucida of the mature ovary, and the expression of amhrII was located in the Sertoli cells of the testis and in the oocytes of the ovary. Decrease in female-related genes and serum 17β-estradiol level, increase in male-related genes and serum 11-ketotestosterone, ovarian regression, and spermatogonia proliferation were observed during plasmid feeding experiment. These results illustrate that amh overexpression plasmid feeding can induce a female-to-male transition in grouper.

Summary Sentence

amh overexpression plasmid feeding can induce a female-to-male transition in grouper.

The recently established immortalized hypothalamic cell model mHypoA-55 possesses characteristics similar to those of Kiss-1 neurons in the arcuate nucleus (ARC) region of the hypothalamus. Here, we show that Kiss-1 gene expression in these cells was downregulated by 17β-estradiol (E2) under certain conditions. Both neurotensin (NT) and corticotropin-releasing hormone (CRH) were expressed in these cells and upregulated by E2. Stimulation of mHypoA-55 cells with NT and CRH significantly decreased Kiss-1 mRNA expression. A mammalian gonadotropin-inhibitory hormone homolog, RFamide-related peptide-3 (RFRP-3), was also found to be expressed inmHypoA-55 cells, and RFRP-3 expression in these cells was increased by exogenous melatonin stimulation. E2 stimulation also upregulated RFRP-3 expression in these cells. Stimulation of mHypoA-55 cells with RFRP-3 significantly increased the expression of NT and CRH. Furthermore, melatonin stimulation resulted in the increase of both NT and CRH mRNA expression in mHypoA-55 cells. On the other hand, in experiments using mHypoA-50 cells, which were originally derived from hypothalamic neurons in the anteroventral periventricular nucleus, Kiss-1 gene expression was upregulated by both NT and CRH, although E2 increased both NT and CRH expression, similarly to the mHypoA-55 cells. Our observations using the hypothalamic ARC cell model mHypoA-55 suggest that NT and CRH have inhibitory effects on Kiss-1 gene expression under the influence of E2 in association with RFRP-3 expression. Thus, these neuropeptides might be involved in E2-induced negative feedback mechanisms.

Summary Sentence

NT, CRH and RFRP-3 might be involved in E2-induced negative feedback mechanisms.

Anti-Müllerian hormone (AMH) helpsmaintain the ovarian reserve by regulating primordial follicle activation and follicular selection inmammals, although its role within the avian ovary is unknown. In mammals, AMH is primarily produced in granulosa cells of preantral and early antral follicles. Similarly, in the hen, the granulosa cells of smaller follicles are the predominant source of AMH. The importance of AMH in mammalian ovarian dynamics suggests the protein and its specific Type II receptor, AMHRII, may have conserved functions in the hen. AMHRII mRNA expression is highest (P < 0.01) in small follicles of the hen and decreases as follicle size increases. Similarly, expression of AMHRII and AMH is highest in granulosa cells from small follicles as compared to larger follicles. Dissection of 3–5 mm follicles into ooplasm and granulosa components shows that AMHRII mRNA levels are greater in ooplasm than granulosa cells. Furthermore, immunohistochemistry also revealed AMHRII staining in the oocyte and granulosa cells. AMH expression in mammals is elevated during periods of reproductive dormancy, possibly protecting the ovarian reserve. AMHRII and AMH mRNA were significantly higher (P < 0.05) in nonlaying ovaries of broiler hens. In molting layer hens, AMHRII mRNA was significantly greater (P < 0.05) compared to nonmolting hen ovaries. These results suggest that AMH may have a direct effect on the oocyte and, thereby, contribute to bidirectional communication between oocyte and granulosa cells. Enhanced expression of AMHRII and AMH during reproductive quiescence supports a potential role of AMH in protecting the ovarian reserve in hens.

Summary SentenceThe expression pattern of anti-Müllerian hormone type II receptor indicates that anti-Müllerian hormone has an important function in the oocyte.

Both oocyte maturation and ovulation is triggered by the luteinizing hormone (LH) surge in vertebrates, but exactly how these processes are regulated by LH remains to be fully elucidated. Previously, we found that Igf3, a fish-specific member of the igf family predominantly expressed in the gonads, could mediate the action of LH on oocyte maturation in zebrafish. Here, we further reveal the importance of Igf3 in mediating the action of LH on ovulation in zebrafish. All the four igf gene family members are expressed in the zebrafish ovary but only the igf3 transcript level is increased in hCG-induced ovulation in vivo. The expression of Igf3 protein in the follicles is also increased during ovulation. The actions of hCG on the expression of ovulatory enzymes and on ovulation itself could be largely mimicked by the recombinant zebrafish Igf3 protein. Intriguingly, the phosphorylation of Igf1r, the receptor for Igf3, could be activated by hCG in the follicular cells during ovulation. And inhibition of Igf3 signaling by Igf1r inhibitors and Igf3 antiserum could significantly attenuate the hCG-induced ovulation. Collectively, all these data support the notion that Igf3 serves as a mediator of LH action in zebrafish ovulation.

Summary Sentence

Igf3 could mediate the action of LH on ovulation in zebrafish.

Establishment of pregnancy in cattle is complex and encompasses ovulation, fertilization, blastocyst formation and growth into an elongated conceptus, pregnancy recognition signaling, and development of the embryo and placenta. The objective here was to investigate sire influences on pregnancy establishment in cattle. First, 10 Holstein bulls were classified as high or low fertility based on their sire conception rate (SCR) value. In a field trial, pregnancy at first timed insemination was not different between high and low SCR bulls. Next, 5 of the 10 sires were phenotyped using in vitro and in vivo embryo production. There was no effect of SCR classification on in vitro embryo cleavage rate, but low SCR sires produced fewer day 8 blastocysts. In superovulated heifers, high SCR bulls produced a lower percentage of unfertilized oocytes and fewer degenerated embryos compared to low SCR bulls. Recipient heifers received three to five in vivo produced embryos from either high or low SCR sires on day 7 postestrus. Day 16 conceptus recovery and length were not different between SCR groups, and the conceptus transcriptome was not appreciably different between high and low SCR sires. The reduced ability of embryos from low SCR bulls to establish pregnancy is multifactorial and encompasses sperm fertilizing ability, preimplantation embryonic development, and development of the embryo and placenta after conceptus elongation and pregnancy recognition. These studies highlight the importance of understanding genetic contributions of the sire to pregnancy establishment that is crucial to increase reproductive efficiency in dairy cattle.

Summary Sentence

The sire influences preimplantation embryonic development and postelongation establishment of pregnancy in dairy cattle.

Maternal pregnancy adaptation is crucial for fetal development and long-term health. Complex interactions occur between maternal digestive and excretory systems as they interface with the developing fetus through the placenta, and transcriptomic regulation in these organs throughout pregnancy is poorly understood. Our objective is to characterize transcriptomic changes across gestation in maternal organs and placenta. Gene expression was quantified in the kidney, liver, and small intestine harvested from nonpregnant and pregnant FVB mice at four time points and placenta at three time points (N = 5/time point) using Affymetrix Mouse Gene 1.0 ST arrays. In maternal organs, we identified 476 genes in the liver, 207 genes in the kidney, and 27 genes in the small intestine that were differentially expressed across gestation (False Discovery Rate [FDR] adjusted q < 0.05). The placenta had a total of 1576 differentially expressed genes between the placenta at either/gd15 or gd19 compared to gd10. We identified a number of pathways enriched for genes differentially expressed across gestation, including 5 pathways in the placenta, 9 pathways in the kidney, and 28 pathways in the liver, including the citrate cycle, retinol metabolism, bile acid synthesis, and steroid bile synthesis, which play functional roles in fetal development and pregnancy maintenance. Characterization of normal longitudinal changes that occur in pregnancy provides context to understand how perturbations in these biochemical pathways and perturbations in nutrient signaling may impact pregnancy.

Summary Sentence

We have characterized differences in metabolic genes and pathways in maternal organs (kidney, liver, small intestine) and placenta of mice over the course of gestation to understand metabolic adaptation to pregnancy.

Caffeine consumption has been widely used as a central nervous system stimulant. Epidemiological studies, however, have suggested that maternal caffeine exposure during pregnancy is associated with increased abnormalities, including decreased fertility, delayed conception, early spontaneous abortions, and low birth weight. The mechanisms underlying the negative outcomes of caffeine consumption, particularly during early pregnancy, remain unclear. In present study, we found that pregnant mice treated with moderate (5 mg/kg) or high (30 mg/kg) dosage of caffeine (intraperitoneally or orally) during preimplantation resulted in retention of early embryos in the oviduct, defective embryonic development, and impaired embryo implantation. Transferring normal blastocysts into the uteri of caffeine-treated pseudopregnant females also showed abnormal embryo implantation, thus indicating impaired uterine receptivity by caffeine administration. The remaining embryos that managed to implant after caffeine treatment also showed increased embryo resorption rate and abnormal development at mid-term stage, and decreased weight at birth. In addition to a dose-dependent effect, significant variations between individual mice under the same caffeine dosage were also observed, suggesting different sensitivities to caffeine, similar to that observed in human populations. Collectively, our data revealed that caffeine exposure during early pregnancy impaired oviductal embryo transport, embryonic development, and uterine receptivity, which are responsible for abnormal implantation and pregnancy loss. The study raises the concern of caffeine consumption during early stages of pregnancy.

Summary Sentence

Caffeine exposure during early pregnancy disrupts oviductal embryo transport, embryonic development and uterine receptivity, leading to aberrant implantation and compromised pregnancy.

Epidemiological studies have revealed that offspring conceived by in vitro fertilization (IVF) have an elevated risk of cardiovascular malformations at birth, and are more predisposed to cardiovascular diseases. The renin-angiotensin system (RAS) plays an essential role in both the pathogenesis of congenital heart disease in fetuses and cardiovascular dysfunction in adults. This study aimed to assess the relative expression levels of genes in the RAS pathway in mice conceived using IVF, compared to natural mating with superovulation. Results demonstrated that expression of the angiotensin II receptor type 1 (AGTR1), connective tissue growth factor (CTGF), and collagen 3 (COL3), in the myocardial tissue of IVF-conceived mice, was elevated at 3 weeks, 10 weeks, and 1.5 years of age, when compared to their non-IVF counterparts. These data were supported by microRNA microarray analysis of the myocardial tissue of aged IVF-conceived mice, where miR-100, miR-297, and miR-758, which interact with COL3, AGTR1, and COL1 respectively, were upregulated when compared to naturally mated mice of the same age. Interestingly, bisulfite sequencing data indicated that IVF-conceived mice exhibited decreased methylation of CpG sites in Col1. In support of our in vivo investigations, miR-297 overexpression was shown to upregulate AGTR1 and CTGF and increased cell proliferation in cultured H9c2 cardiomyocytes. These findings indicate that the altered expression of RAS in myocardial tissue might contribute to cardiovascular malformation and/or dysfunction in IVF-conceived offspring. Furthermore, these cardiovascular abnormalities might be the result of altered DNA methylation and abnormal regulation of microRNAs.

Summary Sentence

Altered expression of the renin-angiotensin system in myocardial tissue might contribute to an increased risk of cardiovascular malformations and dysfunction in IVF offspring, and is involved in abnormal regulations of DNA methylation and microRNAs.

Sertoli cells (SCs) play a central role in testis development, and their normal number and functions are required for spermatogenesis. Although the canonical tuberous sclerosis complex–mammalian target of rapamycin complex 1(TSC-mTORC1) pathway is critical for testis development and spermatogenesis, the signaling mechanisms governing SC functions remain unclear. In this study,we generated two SC-specific mouse mutants using the Cre–LoxP system. Loss of Raptor (a key component of mTORC1) caused severe tubular degeneration in the neonatal testis and adult mice displayed azoospermia, while adult Rheb (an upstream activator for mTORC1) mutant mice had intact tubules and many sperm in their epididymides. Disruption of cytoskeletal organization, including actin, microtubules, and SC-intrinsic vimentin, was observed in Raptor but not Rheb mutant mice. We investigated the reasons for these different effects by whole-transcriptome sequencing, and found that expression of the tight junction adaptor protein cingulin was significantly reduced in Raptor mutant mice. The expression profile of cingulin was synchronous with the differentiation and cytoskeletal dynamics of SCs in control mice, but was disordered in Raptor mutant mice. Furthermore, activity of the small GTPase Rac1 was reduced and expression of the guanine exchange factor for Rac1, Asef, was decreased in Raptor but not Rheb mutant mice. Collectively, these findings establish novel functions of Raptor, independent of the canonical Rheb/mTORC1 pathway, in controlling cytoskeletal homeostasis and cell polarity in SCs, by affecting cingulin expression and Rac1 activity.

Summary Sentence

Raptor has individual functions independence of canonical Rheb/mTOR signaling to regulate cytoskeletal dynamics and cell polarity in Sertoli cells by affecting Cingulin expression and Rac1 activity.

Testicular anti-Müllerian hormone (AMH) production is inhibited by androgens around pubertal onset, as observed under normal physiological conditions and in patients with precocious puberty. In agreement, AMH downregulation is absent in patients with androgen insensitivity. The molecular mechanisms underlying the negative regulation of AMH by androgens remain unknown. Our aim was to elucidate the mechanisms through which androgens downregulate AMH expression in the testis. A direct negative effect of androgens on the transcriptional activity of the AMH promoter was found using luciferase reporter assays in the mouse prepubertal Sertoli cell line SMAT1. A strong inhibition of AMH promoter activity was seen in the presence of both testosterone and DHT and of the androgen receptor. By site-directed mutagenesis and chromatin immunoprecipitation assays, we showed that androgen-mediated inhibition involved the binding sites for steroidogenic factor 1 (SF1) present in the proximal promoter of the AMH gene. In this study, we describe for the first time the mechanism behind AMH inhibition by androgens, as seen in physiological and pathological conditions in males. Inhibition of AMH promoter activity by androgens could be due to protein–protein interactions between the ligand-bound androgen receptor and SF1 or by blockage of SF1 binding to its sites on the AMH promoter.

Summary Sentence

Androgens act directly on prepubertal Sertoli cells to inhibit AMH promoter activity in the presence of the androgen receptor and intact SF1 sites.