Uterine endometrium is one of the most important organs for species preservation. However, the physiology of human endometrium remains poorly understood, because the human endometrium undergoes rapid and large changes during each menstrual cycle and it is very difficult to investigate human endometrium as one organ. This remarkable regenerative capacity of human endometrium strongly suggests the existence of adult stem cells, and physiology of endometrium cannot be explained without adult stem cells. Therefore, investigating endometrial stem/progenitor cells should lead to a breakthrough in understanding the normal endometrial physiology and the pathophysiology of endometrial neoplastic disorders, such as endometriosis and endometrial cancer. Several cell populations have been discovered as putative endometrial stem/progenitor cells. Emerging evidence reveals that the endometrial side population (SP) is one of the potential endometrial stem/progenitor populations. Of all the endometrial stem/progenitor cell candidates, the endometrial SP (ESP) is best investigated in vitro and in vivo, and has the largest number of references. In this review, we provide an overview of the accumulating evidence for the ESP cells, both directly from human endometria and from cultured endometrial cells. Furthermore, SP cells are compared to other potential stem/progenitor cells, and we discuss their stem cell properties. We also discuss the difficulties and unsolved issues in endometrial stem cell biology.

Men are notable for low sperm production, relative to that of other large mammals, and often inferior morphology and motility of their spermatozoa. The extent to which temperature plays a role in this picture has been a moot point. However, animal experiments suggest that an increased scrotal temperature of approximately 4°C brought by inguinal clothing has a negative impact on the germinal epithelium and on the epididymis in man. In two animal species with inguinal testes, their transposition to the abdomen, raising the testis temperature by a modest approximate 1.5°C brought reduced sperm production and abnormalities of spermiogenesis (distorted sperm nuclei, shared acrosomes), a picture seen commonly in man alongside morphologically normal spermatids. Reflection of the scrotal epididymis to the abdomen in laboratory animals did not inhibit sperm maturation there, but the consequences of this for other epididymal parameters are mirrored in several features seen in man. In addition to the typically puny form of the human cauda, these include often rapid epididymal sperm transit, rapid capacitation in vitro, a poor sperm reserve (as reflected in the steep decline in sperm numbers in successive ejaculates), and not least, the cauda's failure to maintain the viability of spermatozoa there (reflected in both their mixed potential for motility and the negative outcome of abstinence). Because the number of competent spermatozoa inseminated relates to prompt fertilization and/or incidence of pregnancy in some animal models, the negative effects of scrotal temperature may be an important factor in the need for an average of approximately five cycles of unprotected intercourse in order to establish pregnancy in human females.

The TGF-beta-SMAD signaling pathway is involved in regulation of various aspects of female reproduction. However, the intrinsic functional role of SMADs in early embryogenesis remains poorly understood. Previously, we demonstrated that treatment with follistatin, an activin (TGF-beta superfamily ligand)-binding protein, is beneficial for bovine early embryogenesis and specific embryotropic actions of follistatin are dependent on SMAD4. Because SMAD4 is a common SMAD that can bind both SMAD2/3 and SMAD1/5, the objective of this study was to further determine the intrinsic role of SMAD2/3 in the control of early embryogenesis and delineate if embryotropic actions of follistatin in early embryos are SMAD2/3 dependent. By using a combination of pharmacological and small interfering RNA-mediated inhibition of SMAD2/3 signaling in the presence or absence of follistatin treatment, our results indicate that SMAD2 and SMAD3 are both required for bovine early embryonic development and stimulatory actions of follistatin on 8- to 16-cell and that blastocyst rates, but not early cleavage, are muted when SMAD2/3 signaling is inhibited. SMAD2 deficiency also results in reduced expression of the bovine trophectoderm cell-specific gene CTGF. In conclusion, the present work provides evidence supporting a functional role of SMAD2/3 in bovine early embryogenesis and that specific stimulatory actions of follistatin are not observed in the absence of SMAD2/3 signaling.

The oocyte-to-embryo transition entails genome activation and a dramatic reprogramming of gene expression that is required for continued development. Superimposed on genome activation and reprogramming is development of a transcriptionally repressive state at the level of chromatin structure. Inducing global histone hyperacetylation relieves this repression and histone deacetylases 1 and 2 (HDAC1 and HDAC2) are involved in establishing the repressive state. Because SIN3A is an HDAC1/2-containing complex, we investigated whether it is involved in reprogramming gene expression during the course of genome activation. We find that Sin3a mRNA is recruited during maturation and that inhibiting its recruitment not only inhibits development beyond the 2-cell stage but also compromises the fidelity of reprogramming gene expression. The SIN3A that is synthesized during oocyte maturation reaches a maximum level in the mid-1-cell embryo and is essentially absent by the mid-2-cell stage. Overexpressing SIN3A in 1-cell embryos has no obvious effect on pre- and postimplantation development. These results provide a mechanism by which reprogramming can occur using a maternally inherited transcription machinery, namely, recruitment of mRNAs encoding transcription factors and chromatin remodelers, such as SIN3A.

Leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is a type of membrane receptor with a seven-transmembrane structure. LGR4 is homologous to gonadotropin receptors, such as follicle-stimulating hormone receptor (Fshr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr). Recently, it has been reported that Lgr4 is a membrane receptor for R-spondin ligands, which mediate Wnt/beta-catenin signaling. Defects of R-spondin homolog (Rspo1) and wingless-type MMTV integration site family, member 4 (Wnt4) cause masculinization of female gonads. We observed that Lgr4^−/− female mice show abnormal development of the Wolffian ducts and somatic cells similar to that in the male gonads. Lgr4^−/− female mice exhibited masculinization similar to that observed in Rspo1-deficient mice. In Lgr4^−/− ovarian somatic cells, the expression levels of lymphoid enhancer-binding factor 1 (Lefl) and Axin2 (Axin2), which are target genes of Wnt/beta-catenin signaling, were lower than they were in wild-type mice. This study suggests that Lgr4 is critical for ovarian somatic cell specialization via the cooperative signaling of Rspo1 and Wnt/beta-catenin.

The dysregulation of endometrial immune response to bacterial lipopolysaccharide (LPS) has been implicated in uterine disease and infertility in the postpartum dairy cow, although the mechanisms are not clear. Here, we investigated whole-transcriptomic gene expression in primary cultures of mixed bovine epithelial and stromal endometrial cells. Cultures were exposed to LPS for 6 h, and cellular response was measured by bovine microarray. Approximately 30% of the 1006 genes altered by LPS were classified as being involved in immune response. Cytokines and chemokines (IL1A, CX3CL1, CXCL2, and CCL5), interferon (IFN)-stimulated genes (RSAD2, MX2, OAS1, ISG15, and BST2), and the acute phase molecule SAA3 were the most up-regulated genes. Ingenuity Pathway Analysis identified up-regulation of many inflammatory cytokines and chemokines, which function to attract immune cells to the endometrium, together with vascular adhesion molecules and matrix metalloproteinases, which can facilitate immune cell migration from the tissue toward the uterine lumen. Increased expression of many IFN-signaling genes, immunoproteasomes, guanylate-binding proteins, and genes involved in the intracellular recognition of pathogens suggests important roles for these molecules in the innate defense against bacterial infections. Our findings confirmed the important role of endometrial cells in uterine innate immunity, whereas the global approach used identified several novel immune response pathways triggered by LPS in the endometrium. Additionally, many genes involved in endometrial response to the conceptus in early pregnancy were also altered by LPS, suggesting one mechanism whereby an ongoing response to infection may interfere with the establishment of pregnancy.

Infection with noncytopathic bovine viral diarrhea virus (ncpBVDV) is associated with uterine disease and infertility. This study investigated the influence of ncpBVDV on immune functions of the bovine endometrium by testing the response to bacterial lipopolysaccharide (LPS). Primary cultures of mixed epithelial and stromal cells were divided into four treatment groups (control [CONT], BVDV, CONT LPS, and BVDV LPS) and infected with ncpBVDV for 4 days followed by treatment with LPS for 6 h. Whole-transcriptomic gene expression was measured followed by Ingenuity Pathway Analysis. Differential expression of 184 genes was found between CONT and BVDV treatments, showing interplay between induction and inhibition of responses. Up-regulation of TLR3, complement, and chemotactic and TRIM factors by ncpBVDV all suggested an ongoing immune response to viral infection. Down-regulation of inflammatory cytokines, chemokines, CXCR4, and serine proteinase inhibitors suggested mechanisms by which ncpBVDV may simultaneously counter the host response. Comparison between BVDV LPS and CONT LPS treatments showed 218 differentially expressed genes. Canonical pathway analysis identified the key importance of interferon signaling. Top down-regulated genes were RSAD2, ISG15, BST2, MX2, OAS1, USP18, IFIT3, IFI27, SAMD9, IFIT1, and DDX58, whereas TRIM56, C3, and OLFML1 were most up-regulated. Many of these genes are also regulated by IFNT during maternal recognition of pregnancy. Many innate immune genes that typically respond to LPS were inhibited by ncpBVDV, including those involved in pathogen recognition, inflammation, interferon response, chemokines, tissue remodeling, cell migration, and cell death/survival. Infection with ncpBVDV can thus compromise immune function and pregnancy recognition, thereby potentially predisposing infected cows to postpartum bacterial endometritis and reduced fertility.

Canine oocyte maturation and fertilization take place within the oviducts under increasing plasma levels of progesterone (P4). In order to investigate the role of P4 in these processes, 51 beagle bitches were treated with the P4 receptor antagonist aglepristone at the end of proestrus and 32 females were kept untreated. Fifteen treated and 13 control bitches were inseminated at Days 1 and 2 after ovulation (Day 0). Stages of oocyte maturation and embryo development were determined after ovariectomy at different time points after ovulation. Aglepristone did not prevent ovulation but delayed the resumption of oocyte meiosis and inhibited its progression: first metaphase I (MI) stage was observed at 173 h postovulation and 39% of oocytes reached MII as late as 335 h postovulation in treated females whereas first MI occurred at 76 h and 100% of oocytes were in MII at 109 h postovulation in controls. Aglepristone extended the stay of morphologically normal oocytes within the oviducts: first signs of oocyte degeneration were observed at 335 h in treated versus 100- to 110-h postovulation in control bitches. In inseminated females, aglepristone prevented sperm progression toward the oviducts and fertilization, although motile spermatozoa were observed in the uterine tip flush and within the cranial uterine glands. A proteomic analysis of the tubal fluid from treated and control noninseminated bitches at Day 4 found evidence of 79 differential proteins potentially involved in the oocyte phenotype. In conclusion, P4 plays key roles in postovulatory canine oocyte maturation, aging, and in fertilization.

Maternal interleukin (IL) 10 deficiency elevates susceptibility to fetal loss induced by the model Toll-like receptor agonist lipopolysaccharide, but the mechanisms are not well elucidated. Here, we show that Il10 null mutant (Il10^−/−) mice exhibit altered local T cell responses in pregnancy, exhibiting pronounced hyperplasia in para-aortic lymph nodes draining the uterus with >6-fold increased CD4 and CD8 T cells compared with wild-type controls. Among these CD4 cells, Foxp3 T regulatory (Treg) cells were substantially enriched, with 11-fold higher numbers at Day 9.5 postcoitum. Lymph node hypertrophy in Il10^−/− mice was associated with more activated phenotypes in dendritic cells and macrophages, with elevated expression of MHCII, scavenger receptor, and CD80. Affymetrix microarray revealed an altered transcriptional profile in Treg cells from pregnant Il10^−/− mice, with elevated expression of Ctse (cathepsin E), Il1r1, Il12rb2, and Ifng. In vitro, Il10^−/− Treg cells showed reduced steady-state Foxp3 expression, and polyclonal stimulation caused greater loss of Foxp3 and reduced capacity to suppress IL17 in CD4 Foxp3⁻ T cells. We conclude that despite a substantially expanded Treg cell pool, the diminished stability of Treg cells, increased numbers of effector T cells, and altered phenotypes in dendritic cells and macrophages in pregnancy all potentially confer vulnerability to inflammation-induced fetal loss in Il10^−/− mice. These findings suggest that IL10 has a pivotal role in facilitating robust immune protection of the fetus from inflammatory challenge and that IL10 deficiency could contribute to human gestational disorders in which altered T cell responses are implicated.

In recent years considerable effort has been devoted to understanding the epigenetic control of sperm development, leading to an increased appreciation of the importance of RNA interference pathways, and in particular miRNAs, as key regulators of spermatogenesis and epididymal maturation. It has also been shown that sperm are endowed with an impressive array of miRNA that have been implicated in various aspects of fertilization and embryo development. However, to date there have been no reports on whether the sperm miRNA signature is static or whether it is influenced by their prolonged maturation within the male reproductive tract. To investigate this phenomenon, we employed next-generation sequencing to systematically profile the miRNA signature of maturing mouse spermatozoa. In so doing we have provided the first evidence for the posttesticular modification of the sperm miRNA profile under normal physiological conditions. Such modifications include the apparent loss and acquisition of an impressive cohort of some 113 and 115 miRNAs, respectively, between the proximal and distal epididymal segments. Interestingly, the majority of these changes occur late in maturation and include the uptake of novel miRNA species in addition to a significant increase in many miRNAs natively expressed in immature sperm. Because sperm are not capable of de novo transcription, these findings identify the epididymis as an important site in establishing the sperm epigenome with the potential to influence the peri-conceptual environment of the female reproductive tract, contribute to the inheritance of acquired characteristics, and/or alter the developmental trajectory of the resulting offspring.

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants for the treatment of depression. However, SSRIs cause sexual side effects such as anorgasmia, erectile dysfunction, and diminished libido that are thought to be mediated through the serotonin (5-hydroxytryptamine, 5-HT) system. In vertebrates, gonadotropin-releasing hormone (GnRH) neurons play an important role in the control of reproduction. To elucidate the neuroendocrine mechanisms of SSRI-induced reproductive failure, we examined the neuronal association between 5-HT and GnRH (GnRH2 and GnRH3) systems in the male zebrafish. Double-label immunofluorescence and confocal laser microscopy followed by three-dimensional construction analysis showed close associations between 5-HT fibers with GnRH3 fibers and preoptic-GnRH3 cell bodies, but there was no association with GnRH2 cell bodies and fibers. Quantitative real-time PCR showed that short-term treatment (2 wk) with low to medium doses (4 and 40 μg/L, respectively) of citalopram significantly decreased mRNA levels of gnrh3, gonadotropins (lhb and fshb) and 5-HT-related genes (tph2 and sert) in the male zebrafish. In addition, short-term citalopram treatment significantly decreased the fluorescence density of 5-HT and GnRH3 fibers compared with controls. Short-term treatment with low, medium, and high (100 μg/L) citalopram doses had no effects on the profiles of different stages of spermatogenesis, while long-term (1 mo) citalopram treatment with medium and high doses significantly inhibited the different stages of spermatogenesis. These results show morphological and functional associations between the 5-HT and the hypophysiotropic GnHR3 system, which involve SSRI-induced reproductive failures.

During embryonic development, mouse female germ cells enter meiosis in an anterior-to-posterior wave believed to be driven by retinoic acid. It has been proposed that ovarian follicle formation and activation follow the same general wave of meiotic progression; however, the precise anatomic specification of these processes has not been delineated. Here, we created a mouse line using Mvh, Gdf9, and Zp3 promoters to drive distinct temporal expression of three fluorescent proteins in the oocytes and to identify where the first follicle cohort develops. The fluorescent profile revealed that the first growing follicles consistently appeared in a specific region of the ovary, the anterior-dorsal region, which led us to analyze if meiotic onset occurred earlier in the dorsal ovarian region. Surprisingly, in addition to the anterior-to-posterior wave, we observed an early meiotic entry in the ventral region of the ovary. This additional anatomic stratification of meiosis contrasts with the localization of the initial follicle formation and activation in the dorsal region of the ovary. Therefore, our study suggests that the specification of cortical and medullar areas in the ventral and dorsal regions on the ovary, rather than the onset of meiosis, impacts where the first follicle activation event occurs.

We previously demonstrated that cigarette smoke (CS) exposure decreases primordial follicle counts and induces autophagy in ovarian granulosa cells in preference to apoptosis. Therefore, the objective of this study was to investigate molecular targets underlying smoke-induced activation of the reparative autophagy pathway in the ovary. Briefly, ovarian homogenates were prepared from adult female mice exposed to mainstream CS twice daily for 8 wk, using a whole-body exposure system. A gene array revealed that CS exposure induced a greater than 2-fold significant increase in the expression of proautophagic genes Cdkn1b, Map1lc3a, Bad, and Sqstm1/p62. A significant increase in Prkaa2, Pik3c3, and Maplc31b expression, as well as a significant decrease in Akt1 and Mtor expression, was detected by quantitative PCR. The 5′-AMP-activated protein kinase catalytic subunit (AMPK) alpha1 alpha2 and ATG7 protein expression was significantly increased, whereas AKT1, mTOR, CDKN1B/p27, and CXCR4 proteins were significantly decreased in CS exposed versus control ovaries. Up-regulation of AMPK alpha1 alpha2, a known initiator of autophagic signaling, and ATG7 further suggests activation of the autophagy cascade. Two prosurvival factors, AKT and mTOR, were decreased in expression, an outcome that favors induction of the autophagy pathway, whereas decreased levels of CDKN1B is suggestive of cell cycle dysregulation. In summary, our data suggest that CS exposure induces ovarian follicle loss through induction of the autophagic cascade via the AMPK pathway together with inhibition of antiautophagic markers AKT and mTOR. We further postulate that toxicant-induced dysregulation of reparative autophagy is a novel pathway central to impaired follicle development and subfertility.

In mammals, follicular atresia can be partially triggered by granulosa cell apoptosis. However, very little is known about the functions of miRNAs in granulosa cell apoptosis. We previously reported that hsa-mir-23a (miR-23a) and hsa-mir-27a (miR-27a) were highly expressed in the plasma of patients with premature ovarian failure, but the action of these two miRNAs in follicular development was unclear. In this study, we explored the roles of miR-23a and miR-27a in the granulosa cells of women undergoing in vitro fertilization/embryo transfer. Using Hoechst staining, we found that miR-23a and miR-27a promoted apoptosis in human granulosa cells. In addition, the Western blotting results suggested that the miR-23a/miR-27a-mediated apoptosis occurred via the FasL-Fas pathway. Based on the results of a luciferase-reporter assay and quantitative RT-PCR and Western blotting analyses, we found that SMAD5 is a target gene of both miR-23a and miR-27a. Furthermore, knocking down SMAD5 expression increased the rate of apoptosis, as well as the levels of Fas, FasL, cleaved caspase-8, and cleaved caspase-3 protein. Taken together, these data suggest that miR-23a and miR-27a target SMAD5 and regulate apoptosis in human granulosa cells via the FasL-Fas pathway. These findings provide an improved understanding of the mechanisms underlying granulosa cell apoptosis, which could potentially be used for future clinical applications.

We studied the smooth muscle cell differentiation capability of human placental multipotent mesenchymal stromal cells (hPMSCs) and identified how endothelial cells recruit hPMSCs participating in vessel formation. hPMSCs from term placentas were induced to differentiate into smooth muscle cells under induction conditions and different matrix substrates. We assessed endothelial cells from umbilical veins for platelet-derived growth factor (PDGF)-BB expression and to induce hPMSC PDGFR-beta and STAT3 activation. Endothelial cells were co-cultured with hPMSCs for in vitro angiogenesis. Cell differentiation ability was then further assessed by mouse placenta transplantation assay. hPMSCs can differentiate into smooth muscle cells; collagen type I and IV or laminin support this differentiation. Endothelial cells expressed significant levels of PDGF-BB and activated STAT3 transcriptional activity in hPMSCs. Endothelial cell-conditioned medium induced hPMSC migration, which was inhibited by STAT3 small interfering RNA transfection or by pretreatement with PDGFR-beta–blocking antibody but not by PDGFR-alpha–blocking antibody or isotype immunoglobulin G (IgG; P < 0.001). hPMSCs can incorporate into endothelial cells with tube formation and promote endothelial cells, forming capillary-like networks than endothelial cells alone (tube lengths: 12 024.1 ± 960.1 vs. 9404.2 ± 584.7 pixels; P < 0.001). Capillary-like networks were significantly reduced by hPMSCs pretreated with PDGFR-beta-blocking antibody but not by PDGFR-alpha–blocking antibody or isotype IgG (P < 0.001). Transplantation of hPMSCs into mouse placentas revealed incorporation of the hPMSCs into vessel walls, which expressed alpha-smooth muscle actin, calponin, and smooth muscle myosin (heavy chain) in vivo. In conclusion, endothelial cell–hPMSC interactions occur during vessel development of placenta. Placental endothelial cell-derived PDGF-BB recruits hPMSCs involved in vascular development via PDGFR-beta/STAT3 activation.

Asynchronous embryo transfer (ET) is a common assisted reproduction technique used in several species, but its biological effects on postnatal and early development remain unknown. The aim of this study was to determine whether asynchronous ET produces long-term effects in mice. Postnatal development, animal weight, systolic blood pressure (SBP), relative organ weight (liver, spleen, kidneys, heart, lungs, brain, and testicles), and behavior (assessed in open-field and elevated plus maze tests) were assessed in CD1 mice produced by different ET procedures: 1) the transfer of Day 3.5 (D3.5) blastocysts to the uterus (BL-UT); 2) the transfer of D3.5 blastocysts to the oviduct (BL-OV); or 3) the transfer of D0.5 zygotes to the oviduct (Z-OV). In vivo conceived animals served as controls (CT). The transfer of blastocysts to the uterus or zygotes to the oviduct was defined as synchronous, and transfer of blastocysts to the oviduct was defined as asynchronous. Both synchronous and asynchronous ET resulted in increased weight at birth that normalized thereafter with the exception of asynchronous ET females. In this group, female BL-OV, a clear lower body weight was recorded along postnatal life when compared with controls (P < 0.05). No effects on animal weight were produced during postnatal development in the synchronous ET groups (BL-UT, Z-OV, and CT). Both synchronous and asynchronous ET had impacts on adult (Wk 30) organ weight. SBP was modified in animals derived from blastocyst but not zygote ET. Effects on behavior (anxiety in the plus maze) were only detected in the BL-UT group (P < 0.05). Our findings indicate that zygotes are less sensitive than blastocysts to ET and that both synchronous and asynchronous blastocyst ET may have long-term consequences on health, with possible impacts on weight, arterial pressure, relative organ weight, and behavior.

Primordial germ cells (PGCs) are the origin of all germ cells in developing embryos. In the sturgeon embryo, PGCs develop from the vegetal hemisphere, which mainly acts as an extraembryonic source of nutrition. Current methods for studying sturgeon PGCs require either killing the fish or using costly and time-consuming histological procedures. Here, we demonstrate that visualization of sterlet (Acipenser ruthenus>) PGCs in vivo is feasible by simply labeling the vegetal hemisphere with fluorescein isothiocyanate (FITC)-dextran. We injected FITC-dextrans, with molecular weights varying between 10 000 and 2 000 000, into the vegetal pole of 1- to 4-cell stage embryos. At the neurula to tail-bud developmental stages, FITC-positive PGC-like cells appeared ventrally around the developing tail bud in the experimental group that received a high-molecular-weight FITC-dextran. The highest average number of FITC-positive PGC-like cells was observed in embryos injected with FITC-dextran having a molecular weight of 500 000 (FD-500). The pattern of migration of the labeled cells was identical to that of PGCs, clearly indicating that the FITC-positive PGC-like cells were PGCs. Labeled vegetal cells, except for the PGCs, were digested and excreted before the embryos starting feeding. FITC-labeled PGCs were observed in the developing gonads of fish for at least 3 mo after injection. We also found that FD-500 could be used to visualize PGCs in other sturgeon species. To the best of our knowledge, this report is the first to demonstrate in any animal species that PGCs can be visualized in vivo for a long period by the injection of a simple reagent.

The spermatozoa of many stallions do not tolerate being cooled, restricting the commercial viability of these animals and necessitating the development of a chemically defined room temperature (RT) storage medium. This study examined the impact of two major modulators of oxidative phosphorylation, pyruvate (Pyr) and L-carnitine (L-C), on the storage of stallion spermatozoa at RT. Optimal concentrations of Pyr (10 mM) and L-C (50 mM) were first identified and these concentrations were then used to investigate the effects of these compounds on sperm functionality and oxidative stress at RT. Mitochondrial and cytosolic reactive oxygen species, along with lipid peroxidation, were all significantly suppressed by the addition of L-C (48 h MitoSOX Red negative: 46.2% vs. 26.1%; 48 and 72 h dihydroethidium negative: 61.6% vs. 43.1% and 64.4% vs. 46.9%, respectively; 48 and 72 h 4-hydroxynonenal negative: 37.1% vs. 23.8% and 41.6% vs. 25.7%, respectively), while the Pyr L-C combination resulted in significantly higher motility compared to the control at 72 h (total motility: 64.2% vs. 39.4%; progressive motility: 34.2% vs. 15.2%). In addition, supplementation with L-C significantly reduced oxidative DNA damage at 72 h (9.0% vs. 15.6%). To investigate the effects of L-C as an osmolyte, comparisons were made between media that were osmotically balanced with NaCl, choline chloride, or L-C. This analysis demonstrated that spermatozoa stored in the L-C balanced medium had significantly higher total motility (55.0% vs. 39.0%), rapid motility (44.0% vs. 25.7%), and ATP levels (70.9 vs. 12.8 ng/ml) following storage compared with the NaCl treatment, while choline chloride did not significantly improve these parameters compared to the control. Finally, mass spectrometry was used to demonstrate that a combination of Pyr and L-C produced significantly higher acetyl-L-carnitine production than any other treatment (6.7 pg/10⁶ spermatozoa vs. control at 4.0 pg/10⁶ spermatozoa). These findings suggest that Pyr and L-C could form the basis of a novel, effective RT storage medium for equine spermatozoa.

The nuclear receptor steroidogenic factor 1 (SF-1, AD4BP, NR5A1) is a key regulator of the endocrine axes and is essential for adrenal and gonad development. Partial rescue of Nr5a1^−/− mice with an SF-1-expressing transgene caused a hypomorphic phenotype that revealed its roles in Leydig cell development. In contrast to controls, all male rescue mice (Nr5a1^−/−;tg ^/0) showed varying signs of androgen deficiency, including spermatogenic arrest, cryptorchidism, and poor virilization. Expression of various Leydig cell markers measured by immunohistochemistry, Western blot analysis, and RT-PCR indicated fetal and adult Leydig cell development were differentially impaired. Whereas fetal Leydig cell development was delayed in Nr5a1^−/−;tg ^/0 embryos, it recovered to control levels by birth. In contrast, Sult1e1, Vcam1, and Hsd3b6 transcript levels in adult rescue testes indicated complete blockage in adult Leydig cell development. In addition, between Postnatal Days 8 and 12, peritubular cells expressing PTCH1, SF-1, and CYP11A1 were observed in control testes but not in rescue testes, indicating SF-1 is needed for either survival or differentiation of adult Leydig cell progenitors. Cultured prepubertal rat peritubular cells also expressed SF-1 and PTCH1, but Cyp11a1 was expressed only after treatment with cAMP and retinoic acid. Together, data show SF-1 is needed for proper development of fetal and adult Leydig cells but with distinct primary functions; in fetal Leydig cells, it regulates differentiation, whereas in adult Leydig cells it regulates progenitor cell formation and/or survival.

To begin embryonic development, the zygote must resume the cell cycle correctly after stimulation by sperm-borne oocyte-activating factors (SOAFs). The postacrosomal WW domain-binding protein (PAWP) is one of the strongest SOAF candidates and is widely conserved among eutherian mammals. It has been reported that the microinjection of recombinant PAWP protein can trigger not only Ca² oscillations in mammalian eggs but also intracellular Ca² release in amphibian eggs. It was also suggested that PAWP is involved in the formation of high-quality spermatozoa. On the other hand, negligible SOAF activity for PAWP cRNA has also been reported. In this study, we generated PAWP null mice and examined the fertilizing ability of male mice. Electron microscopy showed no aberrant morphology in spermatogenesis. Intracytoplasmic injection of a single spermatozoon from the null mouse line showed that depletion of PAWP elicited no quantitative differences in Ca² oscillations or in subsequent development of the embryos. We conclude that PAWP does not play an essential role in mouse fertilization.

Fetal exposure to endocrine disruptors (EDs) is believed to predispose males to reproductive abnormalities. Although males are exposed to combinations of chemicals, few studies have evaluated the effects of ED mixtures at environmentally relevant doses. Our previous work showed that fetal exposure to a mixture of the phytoestrogen genistein (GEN) and the plasticizer di-(2-ethylhexyl) phthalate (DEHP) induced unique alterations in adult testis. In this follow-up study, we examined Postnatal Day 3 (PND3) and PND6 male offspring exposed from Gestational Day 14 to parturition to corn oil, 10mg/kg GEN, DEHP, or their combination, to gain insight into the early molecular events driving long-term alterations. DEHP stimulated the mRNA and protein expression of the steroidogenic enzyme HSD3B, uniquely at PND3. DEHP also increased the mRNA expression of Nestin, a Leydig progenitor/Sertoli cell marker, and markers of Sertoli cell (Wt1), gonocyte (Plzf, Foxo1), and proliferation (Pcna) at PND3, while these genes were unchanged by the mixture. Redox (Nqo1, Sod2, Sod3, Trx, Gst, Cat) and xenobiotic transporter (Abcb1b, Abcg2) gene expression was also increased by DEHP at PND3, while attenuated when combined with GEN, suggesting the involvement of cellular stress in short-term DEHP effects and a protective effect of GEN. The direct effects of GEN and mono-(2-ethylhexyl) phthalate, the principal bioactive metabolite of DEHP, on testis were investigated in PND3 organ cultures, showing a stimulatory effect of 10 μM mono-(2-ethylhexyl) phthalate on basal testosterone production that was normalized by GEN. These effects contrasted with previous reports of androgen suppression and decreased gene expression in perinatal rat testis by high DEHP doses, implying that neonatal effects are not predictive of adult effects. We propose that GEN, through an antioxidant action, normalizes reactive oxygen species-induced neonatal effects of DEHP. The notion that these EDs do not follow classical dose-response effects and involve different mechanisms of toxicity from perinatal ages to adulthood highlights the importance of assessing impacts across a range of doses and ages.