Uterine microbial disease affects half of all dairy cattle after parturition, causing infertility by disrupting uterine and ovarian function. Infection with Escherichia coli, Arcanobacterium pyogenes, and bovine herpesvirus 4 causes endometrial tissue damage. Toll-like receptors on endometrial cells detect pathogen-associated molecules such as bacterial DNA, lipids, and lipopolysaccharide (LPS), leading to secretion of cytokines, chemokines, and antimicrobial peptides. Chemokines attract neutrophils and macrophages to eliminate the bacteria, although persistence of neutrophils is associated with subclinical endometritis and infertility. Cows with uterine infections are less likely to ovulate because they have slower growth of the postpartum dominant follicle in the ovary, lower peripheral plasma estradiol concentrations, and perturbation of hypothalamic and pituitary function. The follicular fluid of animals with endometritis contains LPS, which is detected by the TLR4/CD14/LY96 (MD2) receptor complex on granulosa cells, leading to lower aromatase expression and reduced estradiol secretion. If cows with uterine disease ovulate, the peripheral plasma concentrations of progesterone are lower than those in normal animals. However, luteal phases are often extended in animals with uterine disease, probably because infection switches the endometrial epithelial secretion of prostaglandins from the F series to the E series by a phospholipase A2-mediated mechanism, which would disrupt luteolysis. The regulation of endometrial immunity depends on steroid hormones, somatotrophins, and local regulatory proteins. Advances in knowledge about infection and immunity in the female genital tract should be exploited to develop new therapeutics for uterine disease.

Studies on the reproductive endocrinology of koalas have been performed mainly by using blood samples; however, in practice it is difficult to collect blood periodically because koalas are easily stressed. The purposes of the present study were to establish a noninvasive endocrine monitoring technique and to investigate the reproductive physiology of female koalas. Feces were collected from female northern and southern koalas, and progestagen was extracted from lyophilized fecal samples and determined by enzyme immunoassay. In nonpregnant northern and southern koalas, fecal progestagen markedly increased after copulation and remained high for 36.3 ± 2.5 days and 38.9 ± 1.4 days (luteal phase, mean ± SEM), respectively. Mean (±SEM) progestagen levels (6.34 ± 0.49 μg/g) during the luteal phase in northern koalas were significantly higher than in southern koalas (4.19 ± 0.24 μg/g). Fecal progestagen in parturient northern koalas remained high for 36.2 ± 1.9 days (gestation period, 34.1 ± 0.3 days). In northern koalas, the mean levels and profiles of progestagen during pregnancy (6.44 ± 0.37 μg/g) were consistent with those during nonpregnancy after copulation (6.34 ± 0.49 μg/g). The duration of behavioral estrus in northern koalas was 13.5 ± 0.9 days without copulation. In contrast, when estrous females mated, the estrous sign disappeared just after copulation. The mean (±SEM) length of the estrous cycle in northern koalas, as determined by behavioral estrus intervals, was 33.5 ± 2.2 days without the luteal phase and 69.2 ± 7.6 days with the luteal phase. Fecal progestagen analysis is a helpful and noninvasive tool to monitor ovulatory activity in northern and southern koalas and could help us to understand the reproductive activity of koalas by the combination approach with behavioral estrus.

Amino acids are transported into cells by a number of different transport systems, each with their own specific range of substrates. The amino acid transport systems active in preimplantation embryos and the amino acids required by embryos for optimal development have been extensively investigated. Much less is known about amino acid transport systems active in growing and meiotically maturing oocytes or about developmental changes in their activity. As a first step in determining the array of amino acid transporters active in oocytes, the transport characteristics of nine amino acids were measured in small, medium, and large growing oocytes; in fully grown germinal vesicle (GV)-stage oocytes; in metaphase I oocytes; and in metaphase II eggs. Whether each of 11 classically defined amino acid transport systems was likely active in oocytes at each stage was determined using assays based on measuring the transport of radiolabeled amino acids into oocytes and the effect of a limited set of potential competitive inhibitors. Six amino acid transport systems were found to be active during oocyte growth or maturation. L, b^0, , and ASC/asc were active throughout oocyte growth and maturation, increasing during growth. In contrast, GLY, beta, and x_c⁻ had little or no activity during growth but became activated during meiotic maturation. Surprisingly, the presence of follicular cells surrounding medium growing oocytes or cumulus cells surrounding GV oocytes did not confer amino acid transport by additional transport systems not present in the oocyte. In some cases, however, follicular cells coupled to the oocyte enhanced uptake of amino acids by the same systems present in the oocyte.

In a recently established system for intraperitoneal spermatogonial cell transplantation in salmonids, donor type A spermatogonia (type A SG) were microinjected into the peritoneal cavity of newly hatched larvae. Compared with salmonids, the larvae of marine teleosts are small and vulnerable to physiological and physical stresses, making it difficult to use them for cell manipulation. Herein, we developed type A SG cell transplantation in Nibe croaker (Nibea mitsukurii) by optimizing 1) the developmental stage of the donor testes used to prepare type A SG-enriched cell suspensions and 2) the timing and location of intraperitoneal cell transplantations to recipient larvae. Donor cells labeled with PKH26 fluorescent dye were transplanted into the peritoneal cavity of 3-, 4-, 5-, and 6-mm larvae using glass micropipettes. Consequently, 20.6% of the 4-mm larvae recipients survived for 3 wk, and 36.3% of the survivors had donor-derived cells in their gonads. The incorporated donor cells were identified as germ cells by germ cell-specific nuclear morphology and expression of a germ cell marker. In contrast, no donor type A SG were incorporated into the gonads of 6-mm recipient larvae. These data indicate that there is a distinct narrow window in the developmental stages of recipient larvae when exogenous type A SG can be incorporated into the gonads. The establishment of this system in pelagic egg-spawning marine teleosts would allow the creation of a new broodstock system in which a target species with a large body size and long generation time could be produced from related species with a small body size and short generation time.

Dietary polyunsaturated fatty acids can influence reproductive performance. In dairy cattle, some high-fat diets resulted in higher blastocyst rates and improved embryo quality. These effects may partly be mediated by a direct action of fatty acids on oocyte development. The present study investigated the effect of linolenic acid (ALA; 18:3 n-3) supplementation on bovine oocyte maturation and early embryo development in vitro. Treatment of cumulus-oocyte complexes (COCs) with 50 μM ALA significantly increased the percentage of oocytes at the metaphase II (MII) stage compared with untreated controls (95% ± 2% vs. 84% ± 2%, respectively). Higher doses of ALA were detrimental. Treatment of COCs with 50 μM ALA compared with controls also resulted in a significantly higher percentage of cleaved embryos (77% ± 9% vs. 69% ± 9%, respectively) and blastocyst rate (36% ± 4% vs. 23% ± 5%, respectively) and better-quality embryos. Furthermore, COCs treated with ALA had significant increases compared with controls in: 1) prostaglandin E₂ (PGE₂) concentration (233% ± 41%) in the medium, 2) intracellular cAMP at 3 h of maturation, and 3) phosphorylation of the mitogen-activated protein kinases (MAPKs) during the first 6 h of maturation. Moreover, ALA overcame the suppressive effects of the prostaglandin-endoperoxide synthase 2 inhibitor (NS-398) on oocyte maturation and partially improved the maturation rate in the presence of the MAPK kinase inhibitor (U-0126). Linolenic acid could not, however, recover maturation in the presence of both inhibitors. In conclusion, treatment of bovine COCs with ALA during oocyte maturation affects the molecular mechanisms controlling oocyte nuclear maturation, leading to an increased number of MII-stage oocytes and improved subsequent early embryo development. This effect is mediated both directly through MAPK pathway and indirectly through PGE₂ synthesis.

The protandrous black porgy, Acanthopagrus schlegeli, has a striking life cycle with sex differentiation at the juvenile stage, mono-male development, and male-to-female sex change (with vitellogenic oocytes) at age 3 yr. In the present study, we investigated the possible roles of wnt4 in gonadal development in a nonmammalian model organism (protandrous black porgy), especially in relation to sex differentiation, ovarian growth, and sex change. Fish of various ages were treated with estradiol (E2) or aromatase inhibitor (AI) to determine whether manipulation of the hormonal environment had an effect on these processes. Furthermore, a natural sex change (≥2-yr-old fish) and a nonchemical method to induce an early sex change (≥1-yr-old fish) via the removal of testicular tissue were examined in this study. We present herein an integrative immunohistochemical, cellular, and molecular data set describing these phenomena. During gonadal sex differentiation, no increase in wnt4 expression was detected. A profile of increased wnt4 expression and decreased cyp19a1a expression was associated with ovarian growth (proliferation of oogonia and development of ovarian lamellae) in ≥1-yr-old fish. Both E2 and AI induced an increase in wnt4 transcripts and resulted in ovarian development in ≥0-yr-old and ≥1-yr-old fish. Increased wnt4 transcripts were found in ovarian tissue undergoing development from primary oocytes to vitellogenic oocytes during the natural sex change in ≥2-yr-old fish. Removal of testicular tissue in ≥1-yr-old fish resulted in successful early sex change (with vitellogenic oocytes) 6 mo after the excision. During the process of the early sex change (3 mo after testis excision), the fish ovary became active and had increased diameter of the primary oocytes; this was in accord with increased ovarian wnt4 expression but not sf1, foxl2, and genes in the steroidogenic pathway, including cyp19a1a. Wnt4 staining further confirmed the profile of wnt4 expression associated with ovarian development. The results of the present study suggest that wnt4 has important roles in late ovarian growth (e.g., oogonia proliferation and structure of ovarian lamellae) and the natural sex change (vitellogenic oocytes) in the protandrous black porgy.

Cluster analysis at Postnatal Day 8–20 of putative androgen-regulated genes in mice with Sertoli cell-selective knockout of the androgen receptor (SCARKO) has pinpointed three genes (Spinlw1, Gpd1, Drd4) with an expression pattern strongly resembling that of Rhox5, the definitive Sertoli cell (SC) androgen-regulated gene. We used organotypic testis cultures from Day 8 mice to study control of these genes by (anti)androgens and follicle-stimulating hormone (FSH). Testis morphology and androgen induction of the studied genes were preserved for 48 h. Preincubation with ketoconazole for 24 h to block endogenous androgen production, followed by 24-h incubation with the synthetic androgen R1881, resulted in 45-, 5-, 19-, and 6-fold induction of mRNA levels of Rhox5, Spinlw1, Gpd1, and Drd4, respectively. However, noticeable differences in control of the studied genes were observed. Rhox5 and Spinlw1 were fully induced by R1881 in the continuous (48 h) presence of ketoconazole, whereas only marginal effects were observed on expression of Gpd1 and Drd4. Similarly, FSH only marginally affected expression of Rhox5 and Spinlw1, whereas it markedly increased Gpd1 and Drd4 expression. Explant cultures of SCARKO testes confirmed the differential effects of FSH on the studied genes and, for Gpd1, showed that the effect did not depend on a functional androgen receptor in SC, whereas this was essential for the effects of FSH on Drd4. In conclusion, organotypic cultures represent the first in vitro approach to preserving androgen responsiveness of putative SC-expressed genes. This approach facilitates detailed analysis of their regulation in ways not possible in vivo.

Reduced hypothalamic sensitivity to steroid negative feedback may contribute to the onset of puberty. In high fat-fed rodents, the timing of vaginal opening (VO) is advanced, suggesting that puberty begins earlier. Because obesity can increase androgens, which interfere with normal steroid feedback in adult females, we hypothesized that androgens reduce hypothalamic sensitivity to negative feedback during puberty and that blocking androgen action would prevent advanced VO in high fat-fed mice. Age at VO was examined in mice fed high-fat or low-fat diets from weaning and treated with the androgen receptor antagonist flutamide or vehicle (controls). VO was advanced in high-fat vs. low-fat controls, and flutamide blocked this advancement. VO was also delayed in low fat-fed flutamide-treated females, suggesting involvement of androgens in the timing of normal puberty. We next investigated if high-fat diet-induced insulin resistance contributes to early VO, as elevated insulin can stimulate androgen production. VO was examined in mice on either diet treated with the insulin sensitizer metformin. Metformin blocked high-fat advancement of VO but did not alter the timing of VO in low fat-fed mice. Insulin was elevated in high fat-fed females that had undergone VO compared with age-matched low fat-fed or metformin-treated animals on either diet that had not undergone VO. Together, these data suggest a model in which metabolic changes induced by high-fat diet, including transient increased circulating insulin, act in part by increasing androgen action to influence the timing of puberty in females.

High doses of the commonly used herbicide atrazine have been shown to suppress luteinizing hormone (LH) release. To determine whether atrazine alters the function of gonadotropin-releasing hormone (GnRH) neurons, we examined the effects of atrazine on GnRH neuronal activation and the subsequent release of LH normally associated with ovulation. Ovariectomized adult Wistar rats were administered atrazine (50, 100, or 200 mg/kg of body weight daily by gavage) or vehicle for 4 days. Animals were primed with estrogen and progesterone to induce an evening LH surge. Blood samples were obtained over the afternoon and evening using an indwelling right atrial cannula, and plasma was assayed for LH and FSH. Another cohort of animals was transcardially perfused in the afternoon to examine GnRH activation using FOS immunoreactivity. Results of these studies show that 4-day treatment with atrazine resulted in a significant reduction in the magnitude of the LH and FSH surges, and this corresponds to a decrease in GnRH neurons expressing FOS immunoreactivity. To determine if the effects of atrazine were long lasting, additional studies were performed examining LH levels and GnRH activation 2 days and 4 days after atrazine withdrawal. Within 4 days (but not 2 days) after cessation of atrazine treatment, measures of hypothalamic-pituitary-gonadal (HPG) activation returned to normal. These data indicate that atrazine affects neuroendocrine function in the female rat by actions at the level of the GnRH neuron and that the acute effects of high doses of atrazine can be reversed within 4 days after withdrawal of treatment.

The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an apparent molecular mass of approximately 83 kDa. On the other hand, the Ab developed against the NOS3 showed a band of approximately 96 kDa in fresh and FT sperm lysates. NO production was positively correlated with sperm motility and velocity after thaw, suggesting an NO role for the functionality of cryopreserved stallion spermatozoa; but the production of NO is compromised in egg yolk-containing extenders.

The ruminant conceptus undergoes a period of elongation that is required for maternal recognition of pregnancy, prior to attaching to the endometrium. The purpose of these studies was to investigate the role of proline-rich 15 (PRR15) in the sheep conceptus by examining mRNA expression, protein localization, and the effect of PRR15 mRNA degradation. Conceptuses were collected on Days 11, 13, 15, 16, 17, 21, and 30 after mating. Quantitative RT-PCR showed expression of PRR15 mRNA corresponded with the process of trophoblast elongation, with peak expression occurring on Days 15 and 16. A recombinant ovine PRR15 was generated and used to create polyclonal antibodies against PRR15. Immunohistochemistry of a Day 15 conceptus indicated that PRR15 was localized predominantly in the nucleus of the trophectoderm and extraembryonic primitive endoderm. To test whether PRR15 was required during early conceptus development, RNA interference was employed. Blastocysts collected on Day 8 after mating were infected with a lentivirus expressing a short-hairpin RNA (shRNA) that targeted PRR15 mRNA for degradation, an shRNA containing a three-nucleotide mismatch to PRR15 mRNA, or a lentivirus expressing no shRNA. After infection, blastocysts were transferred into recipient ewes and collected back on Day 15 of gestation. Although the majority of the control and mismatched shRNA-treated conceptuses elongated and survived to Day 15, none of the embryos treated with the lentivirus expressing shRNA against PRR15 mRNA elongated, and most died. In conclusion, expression of PRR15 mRNA occurred during a narrow window of conceptus development, and degradation of PRR15 mRNA led to conceptus demise or abnormal development.

Calcium ions have been implicated in the establishment and maintenance of pregnancy, but the regulatory mechanisms of calcium ions in the uterine endometrium and conceptus are not well understood in pigs. Recently, we showed that TRPV6, a calcium ion channel protein associated with cellular entry of calcium ions, is highly expressed in the uterine endometrium during the implantation period in pigs. In the present study, we investigated spatial and temporal expression and regulation of TRPV6 and S100G, an intracellular calcium-regulatory molecule, in the uterine endometrium during the estrous cycle and pregnancy in pigs. TRPV6 expression was maintained at significantly higher levels in the uterine endometrium during pregnancy compared with levels during the estrous cycle. TRPV6 transcripts and proteins were localized mainly to luminal epithelial cells (LE) and weakly to glandular epithelial cells (GE) and chorionic membrane (CM) during pregnancy. TRPV6 expression was also detected in conceptuses on Day (D) 12 and D15. TRPV6 mRNA levels in the endometrium were increased by estrogen treatment. S100G expression showed a biphasic pattern of increases on D12 of pregnancy and from D60 to term pregnancy, and it localized primarily to LE during early pregnancy and to LE, GE, and CM from D30 to term pregnancy. These results indicate that spatial and temporal expression of TRPV6 and S100G is dynamically regulated in the uterine endometrium during pregnancy and that endometrial regulation of calcium ion concentration by TRPV6 and S100G may be critical for the establishment and maintenance of pregnancy in pigs.

Cytosolic phospholipase A2 (cPLA2, PLA2G4A) catalyzes the release of arachidonic acid for prostaglandin synthesis by cyclooxygenase 1 (PTGS1) and cyclooxygenase 2 (PTGS2). Mice with Pla2g4a deficiency have parturition delay and other reproductive deficits, including deferred onset of implantation, crowding of implantation sites, and small litters. In this study, we examined the contribution of PLA2G4A to parturition in mice. Pla2g4a mRNA and protein expression were discretely localized in the term and preterm uterine luminal epithelium and colocalized with Ptgs1, but not Ptgs2, expression. The levels of PGE2, PGF2alpha, 6-keto-PGF1alpha, and TxB2 were significantly decreased in Pla2g4a-null uterine tissues, similar to Ptgs1-null uteri, consistent with predominance of PLA2G4A-PTGS1-mediated prostaglandin synthesis in preparation for murine parturition. Litter size was strongly associated with the timing of parturition in Pla2g4a-null mice but could not fully account for the parturition delay. Pla2g4a-null females that received PGE2 carbaprostacyclin at the time of implantation delivered earlier (20.5 ± 0.2 days vs. 21.6 ± 0.2 days, P < 0.01), although litter size was not improved (4.6 vs. 4.4 pups per litter, P = 0.6). After correction for small litter size, multivariate analysis indicated that Pla2g4a-null mice given prostaglandin treatment to improve implantation timing had gestational length that was similar to wild-type and Pla2g4a heterozygous mice. These results indicate that, despite specific Pla2g4a expression and function in term gestation uteri, the delayed parturition phenotype in Pla2g4a-null mice is primarily due to deferral of implantation. The role of PLA2G4A in timely parturition appears to be critically related to its actions in early pregnancy.

There is a vital need to identify factors that enhance human and nonhuman primate in vitro embryo culture and outcome, and to identify the factors that facilitate that objective. Granulosa and cumulus cells were obtained from rhesus monkeys that had either been FSH-primed (in vitro maturation [IVM]) or FSH and hCG-primed (in vivo maturation [VVM]) and compared for the expression of mRNAs encoding follistatin (FST), inhibin, and activin receptors. The FST mRNA displayed marginally decreased expression (P = 0.05) in association with IVM in the granulosa cells. The ACVR1B mRNA was more highly expressed in cumulus cells with IVM compared with VVM. Cumulus-oocyte complexes from FSH-primed monkeys exposed to exogenous FST during the 24-h IVM period exhibited no differences in the percentage of oocytes maturing to the metaphase II stage of meiosis compared to controls. However, embryos from these oocytes had significantly decreased development to the blastocyst stage. The effect of FST on early embryo culture was determined by exposing fertilized VVM oocytes to exogenous FST from 12 to 60 h postinsemination. FST significantly improved time to first cleavage and embryo development to the blastocyst stage compared with controls. The differential effects of exogenous FST on embryo development, when administered before and after oocyte maturation, may depend on the endogenous concentration in cumulus cells and oocytes. These results reveal evolutionary conservation of a positive effect of FST on embryogenesis that may be broadly applicable to enhance in vitro embryogenesis, with potential application to human clinical outcome and livestock and conservation biology.

The nonobese diabetic (NOD) mouse is a valuable model for human type 1 diabetes and the development of humanized mice. Although the importance of this mouse strain is widely recognized, its usefulness is constrained by the absence of NOD embryonic stem (ES) lines with adequate germline transmission competence. In the present study, we established two germline transmission-competent types of cell lines from NOD mice; these cell lines, male germline stem (GS) cells and ES cells, were derived from NOD spermatogonia and blastocysts, respectively. NOD-GS cells proliferated in vitro and differentiated into mature sperm after transplantation into testis. NOD-ES cell lines were effectively established from NOD blastocysts using culture medium containing inhibitors for fibroblast growth receptor, MEK, and GSK3. Both the NOD-GS and NOD-ES cell lines transmitted their haplotypes to progeny, revealing a novel strategy for gene modification in a pure NOD genetic background. Our results also suggest that the establishment of GS cells is an effective procedure in nonpermissive mouse strains or other species for ES cell derivation.

Psychosocial factors, particularly social stress, may compromise reproduction. However, some individuals may be more susceptible to socially induced infertility. The present study used group-housed, adult, ovariectomized rhesus monkeys to test the hypothesis that exposure to psychosocial stress, imposed by social subordination, would enhance estradiol (E2)-negative feedback inhibition of LH. Because polymorphisms in the gene encoding the serotonin transporter (SLC6A4) may contribute to individual differences in response to adverse environments, we determined whether subordinate females with the short-promoter-length allele (s-variant) would show greater suppression of LH. Subordinate females, particularly those with the s-variant SLC6A4 genotype, received significantly higher rates of noncontact aggression from more dominant cage mates and had consistently lower body weights. Serum LH was not influenced by social status in the absence of E2. In contrast, subordinate females were hypersensitive to E2-negative feedback inhibition of LH. Furthermore, serum LH in subordinate females with s-variant SLC6A4 genotype was maximally suppressed by Day 4 of treatment, whereas nadir concentrations were not reached until later in treatment in other females. Finally, pharmacological elevation of serum cortisol potentiated E2-negative feedback inhibition in all females. The current data suggest that infertility induced by psychosocial stressors may be mediated by hypersensitivity to E2-negative feedback and that polymorphisms in the SLC6A4 gene may contribute to differences in reproductive compromise in response to chronic stress.

The reproductive potential of mammals decreases with aging, until reaching infertility. One reason for aging-related infertility is the decrease of the reproductive capability of old oocytes. It was found previously that gene expression, histone acetylation, and protein function are altered by aging in metaphase II (MII) stage oocytes. MII oocytes develop from germinal vesicle (GV)-stage oocytes. Here, we hypothesized that the defects of old MII oocytes arise at the GV stage. To prove this hypothesis, we examined the acetylations of histone H4 at lysines 5 (H4K5), 8 (H4K8), 12 (H4K12), and 16 (H4K16) in old GV and MII oocytes. We found that acetylation of H4K12 and H4K16 decreased in old GV oocytes. Acetylation of H4K12 later increased in old MII oocytes. We also examined expression of Cdc2a, a gene related to H4K12 acetylation. Cdc2a expression increased in old nonsurrounded nucleolus (NSN) oocytes but decreased in old MII oocytes. On the other hand, the protein and kinase activities of CDC2A decreased in both GV and MII old oocytes. Finally, we showed that correction of the histone deacetylation of old oocytes at the GV stage restores younglike levels of H4K12 acetylation and CDC2A protein at the MII stage. These data support our hypothesis that abnormalities of histone acetylation at the GV stage are the cause of alterations at the MII stage. Our study provides evidence for strategies targeting the GV stage of oocytes to overcome aging-induced infertility.

Embryo-induced signaling pathways are considered to be important for initiation and sustenance of pregnancy. However many of these pathways remain to be deciphered in primates. In the present study, differential display RT-PCR was used to identify genes or gene fragments that are differentially expressed in endometrium of bonnet monkeys (Macaca radiata) on Day 6 of pregnancy. Of several fragments found to be differentially expressed, a fragment of 567 base pair (named GG1) was characterized in detail. GG1 was highly represented in endometrium of pregnant animals compared with that of nonpregnant animals. Sequencing analysis revealed homology of this fragment to exons 7, 8, 9, and 10 and surprisingly to intron 6 of cAMP-dependent protein kinase A (PKA) regulatory type I alpha (tissue-specific extinguisher 1) (PRKAR1A). The increased expression of this fragment in gestational endometrium was confirmed by quantitative PCR studies. Two transcripts of 3.0 kilobase (kb) and 1.5 kb were detected in Northern blot probed with labeled GG1. Protein expressions of alpha regulatory (PRKAR1A) and alpha catalytic (PRKCA) subunits of PKA were also higher in gestational endometrium compared with that in nongestational endometrium. Further in vitro studies using human endometrial explants demonstrated regulation of PRKAR1A (or GG1) and prostaglandin-endoperoxide synthase 2 or cyclooxygenase 2 (PTGS2) by estradiol. This is the first study to date on the differential expression of PKA in primate endometrium during early pregnancy and its in vitro regulation by estradiol.

Embryo implantation involves direct interaction of the blastocyst with the luminal epithelium of the receptive uterus. MUC1, a transmembrane mucin expressed at the apical surface of uterine epithelia, acts as a barrier to microbial infection and enzymatic attack. Loss of MUC1 is believed to be a prerequisite for a functionally receptive uterus across many species. Human and murine MUC1 regulation by steroid hormones displays important differences. Estrogen (E2) stimulates MUC1 expression in mice, and progesterone (P4) antagonizes E2 action in this regard. MUC1 expression is severely reduced during the receptive uterine state in mice. In contrast, human MUC1 expression is maximal at the receptive or midluteal phase, when P4 levels are high. No information is available regarding regulation of human MUC1 in vivo at the site of embryo attachment. Our aim was to better understand regulation of human MUC1 during early pregnancy in vivo. For this purpose, we used a transgenic mouse carrying full-length human MUC1 gene (Tg(MUC1)79.24Gend) as well as endogenous MUC1 as a model system. Human MUC1 was detected by real-time RT-PCR, Western blotting, and immunohistochemistry during early pregnancy. Our data indicate that human MUC1 persists at reduced (20% relative to Day 1 postcoitum) levels in receptive-phase uteri, including the site of embryo attachment. In contrast, mouse MUC1 was much more severely (>98% relative to Day 1 postcoitum) reduced in the same context. These observations are consistent with distinct regulation between the human and mouse genes. Because these genes are expressed in the same transcriptional context (i.e., mouse uterine epithelia), structural differences between human and murine genes must account for these differences in MUC1 regulation.

The study of alternative model organisms has yielded tremendous insights into the regulation of behavioral and physiological traits not displayed by more widely used animal models, such as laboratory rats and mice. In particular, comparative approaches often exploit species ideally suited for investigating specific phenomenon. For instance, comparative studies of socially monogamous prairie voles and polygamous meadow voles have been instrumental toward gaining an understanding of the genetic and neurobiological basis of social bonding. However, laboratory studies of less commonly used organisms, such as prairie voles, have been limited by a lack of genetic tools, including the ability to manipulate the genome. Here, we show that lentiviral vector-mediated transgenesis is a rapid and efficient approach for creating germline transgenics in alternative laboratory rodents. Injection of a green fluorescent protein (GFP)-expressing lentiviral vector into the perivitelline space of 23 single-cell embryos yielded three live offspring (13 %), one of which (33%) contained germline integration of a GFP transgene driven by the human ubiquitin-C promoter. In comparison, transfer of 23 uninjected embryos yielded six live offspring (26%). Green fluorescent protein is present in all tissues examined and is expressed widely in the brain. The GFP transgene is heritable and stably expressed until at least the F(2) generation. This technology has the potential to allow investigation of specific gene candidates in prairie voles and provides a general protocol to pursue germline transgenic manipulation in many different rodent species.

The hypodactylous (hd) locus impairs limb development and spermatogenesis, leading to male infertility in rats. We show that the hd mutation is caused by an insertion of an endogenous retrovirus into intron 10 of the Cntrob gene. The retroviral insertion in hd mutant rats disrupts the normal splicing of Cntrob transcripts and results in the expression of a truncated protein. During the final phase of spermiogenesis, centrobin localizes to the manchette, centrosome, and the marginal ring of the spermatid acroplaxome, where it interacts with keratin 5-containing intermediate filaments. Mutant spermatids show a defective acroplaxome marginal ring and separation of the centrosome from its normal attachment site of the nucleus. This separation correlates with a disruption of head-tail coupling apparatus, leading to spermatid decapitation during the final step of spermiogenesis and the absence of sperm in the epididymis. Cntrob may represent a novel candidate gene for presently unexplained hereditary forms of teratozoospermia and the “easily decapitated sperm syndrome” in humans.

Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower frequencies favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1 and its two corepressors, Ngfi-A-binding proteins 1 and 2 (NAB1 and NAB2), both in vivo and in cultured rat pituitary cells. In rats, fast GNRH pulses (every 30 min) stably induced Egr1 primary transcript (PT) and mRNA 2-fold (P < 0.05) for 1–24 h. In contrast, slow GNRH pulses (every 240 min) increased Egr1 PT at 24 h (6-fold; P < 0.05) but increased Egr1 mRNA 4- to 5-fold between 4 and 24 h. Both GNRH pulse frequencies increased EGR1 protein 3- to 4-fold. In cultured rat pituitary cells, GNRH pulses (every 60 min) increased Egr1 (PT, 2.5- to 3-fold; mRNA, 1.5- to 2-fold; P < 0.05). GNRH pulses had little effect on Nab1/2 PT/mRNAs either in vivo or in vitro. We also examined specific intracellular signaling cascades activated by GNRH. Inhibitors of mitogen-activated protein kinase 8/9 (MAPK8/9 [also known as JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [also known as MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (also known as p38) inhibitor SB203580 did not. In summary, pulsatile GNRH stimulates Egr1 gene expression and protein in vivo but not in a frequency-dependent manner. Additionally, GNRH-induced Egr1 gene expression is mediated by MAPK8/9 and MAPK1/3, and both are critical for Lhb gene transcription.

The brain mechanism regulating gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release is sexually differentiated in rodents. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) have been suggested to be sexually dimorphic and involved in the GnRH/LH surge generation. The present study aimed to determine the significance of neonatal testicular androgen to defeminize AVPV kisspeptin expression and the GnRH/LH surge-generating system. To this end, we tested whether neonatal castration feminizes AVPV kisspeptin neurons and the LH surge-generating system in male rats and whether neonatal estradiol benzoate (EB) treatment suppresses the kisspeptin expression and the LH surge in female rats. Immunohistochemistry, in situ hybridization, and quantitative real-time RT-PCR were performed to investigate kisspeptin and Kiss1 mRNA expressions. Male rats were castrated immediately after birth, and females were treated with EB on postnatal Day 5. Neonatal castration caused an increase in AVPV kisspeptin expression at peptide and mRNA levels in the genetically male rats, and the animals showed surge-like LH release in the presence of the preovulatory level of estradiol (E2) at adulthood. On the other hand, neonatal EB treatment decreased the number of AVPV kisspeptin neurons and caused an absence of E2-induced LH surge in female rats. Semiquantitative RT-PCR analysis showed that neonatal steroidal manipulation affects Kiss1 expression but does not significantly affect gene expressions of neuropeptides (neurotensin and galanin) and enzymes or transporter for neurotransmitters (gamma-aminobutyric acid, glutamate, and dopamine) in the AVPV, suggesting that the manipulation specifically affects Kiss1 expressions. Taken together, our present results provide physiological evidence that neonatal testicular androgen causes the reduction of AVPV kisspeptin expression and failure of LH surge in genetically male rats. Thus, it is plausible that perinatal testicular androgen causes defeminization of the AVPV kisspeptin system, resulting in the loss of the surge system in male rats.

Premature cervical ripening is believed to contribute to preterm birth (PTB). Preterm cervical ripening may be due to an aberrant regulation in timing of the same processes that occur at term, or may result from unique molecular mechanisms. Using mouse models of PTB, this study sought to investigate if the molecular mechanisms that govern cervical ripening were similar between preterm and term. Lipopolysaccharide (LPS) is infused into the uterine horn to create a mouse model of inflammation-induced PTB. For a noninfectious model of PTB, RU486 was administered. Both models result in delivery of pups in 8–24 h. Cervical tissues were collected from these models, as well as throughout gestation. Cervical tissues from E15 (preterm), E15 LPS (preterm inflammation), and E18.5 (term) were used for microarray analysis (n = 18). Additional experiments using gestational time course specimens were performed to confirm microarray results. Specific gene pathways were differentially expressed between the groups. Genes involved in immunity and inflammation were increased in the cervix in inflammation-induced PTB; term labor was not associated with differential expression of immune pathways. Cytokine expression was not increased in cervices during term labor, but was increased in the pospartum period. Epithelial cell differentiation pathway was significantly altered in term, but not preterm, labor. Activation of immune pathways may be sufficient for cervial ripening, but does not appear necessary. Differential expression of the epithelial cell differentiation pathway appears necessary in the process of cervical repair. Our results indicate that the molecular mechanisms governing preterm and term cervical ripening are distinctly different.