Mouse blastocyst outgrowth in vitro and probably implantation in vivo require amino acid signaling via the target of rapamycin (TOR) pathway. This signaling does not simply support protein synthesis and trophoblast differentiation. Rather, it regulates development of trophoblast protrusive activity and may act as a developmental checkpoint for implantation. Moreover, intracellular amino acids per se are insufficient to elicit TOR signaling. Instead, de novo transport of amino acids, and particularly of leucine, stimulate mTOR activity at the blastocyst stage. The activity of the broad-scope and yet leucine-selective amino acid transport system B^0, could produce such increases in intracellular amino acid concentrations. For example, system B^0, uses a Na gradient to drive amino acid uptake, and the Na concentration in uterine secretions increases by nearly two-fold about 18 h before implantation. The resultant mTOR signaling could trigger polyamine, insulin-like growth factor II, and nitric oxide production in blastocysts and the increased cell motility sometimes associated with synthesis of these bioactive molecules.

The presence of ammonium in the culture medium has significant detrimental effects on the regulation of embryo physiology and genetics. Ammonium levels build up linearly over time in the culture medium when media containing amino acids are incubated at 37°C. Ammonium in the culture media significantly reduces blastocyst cell number, decreases inner cell mass development, increases apoptosis, perturbs metabolism, impairs the ability of embryos to regulate intracellular pH, and alters the expression of the imprinted gene H19. In contrast, the rate of blastocyst development and blastocyst morphology appear to be normal. The transfer of blastocysts exposed to ammonium results in a significant reduction in the ability to establish a pregnancy. Furthermore, of those embryos that manage to implant, fetal growth is significantly impaired. Embryos exposed to 300 μM ammonium are retarded by 1.5 days developmentally at Day 15 of pregnancy. It is therefore essential that culture conditions for mammalian embryos are designed to minimize the buildup of ammonium to prevent abnormalities in embryo physiology, genetic regulation, pregnancy, and fetal development.

Female macaques produced isoantibodies to a limited number of sperm surface proteins following immunization with sperm components released by phosphatidylinositol-specific phospholipase C (PI-PLC). Washed, acrosome-intact, fixed sperm injected into rabbits elicited a major immune response to one of the same PI-PLC-released proteins, which was shown to be a sperm surface-coating protein. After purification and digestion of the glycoprotein, four peptides were analyzed for amino acid sequence, and all had 100% homology with an epididymal secretory protein, ESP13.2, reported previously to be a small, cationic-rich peptide and a member of the β-defensin family. Antibodies to purified ESP13.2 recognized a number of protein bands on Western blots of nonreduced PI-PLC-released sperm components and nonreduced whole-sperm extracts. After chemical disulfide reduction, only a single, broad band from 31 to 35 kDa was recognized by anti-ESP13.2 antibodies. Indirect immunofluorescence showed ESP13.2 over the entire surface of ejaculated macaque sperm. Fluorescence was only slightly reduced after sperm were washed through 80% Percoll. A 24-h incubation in capacitating medium significantly reduced the amount of ESP13.2 over the head and midpiece, whereas exposure of the incubated sperm to dbcAMP and caffeine (capacitation activators) resulted in almost complete loss of ESP13.2 from the sperm surface. After activation, ESP13.2 was the primary component released into the medium as judged electrophoretically. Lignosulfonic acid, a potent inhibitor of macaque fertilization in vitro, completely blocked release of ESP13.2 from the sperm surface, even following treatment with activators. These findings suggest that the β-defensin, ESP13.2, has a function in the capacitation of macaque spermatozoa and may modulate sperm surface-receptor presentation at the time of fertilization.

Hyperhomocysteinemia has been suggested as a possible risk factor in women suffering from habitual abortions, placental abruption or infarcts, preeclampsia, and/or intrauterine growth retardation. However, little is known about the pathogenic mechanisms underlying the action of homocysteine. The present study investigated the in vitro ability of homocysteine to affect trophoblast gonadotropin secretion and to induce cell death. In primary human trophoblast cells, homocysteine treatment (20 μmol/L) resulted in cellular flattening and enlargement, extension of pseudopodia, and cellular vacuolization. Cellular detachment, apoptosis, and necrosis were favored. With in situ nick end labeling, we investigated DNA degradation, and we used M30 CytoDEATH to selectively stain the cytoplasm of apoptotic cells. Cytochrome c release from mitochondria to the cytosol and DNA cleavage in agarose gel have been investigated. Homocysteine, but not cysteine, induced trophoblast apoptosis and significantly reduced human chorionic gonadotropin secretion. These findings suggest that trophoblast cell death might represent a pathogenic mechanism by which homocysteine may cause pregnancy complications related to placental diseases.

Analysis by cDNA microarrays showed that in the murine epididymis, NaP_i-IIb was the predominantly expressed epithelial isoform of the sodium-inorganic phosphate cotransporter and was markedly overexpressed in the proximal region in the infertile knockout (KO) compared to the fertile heterozygous (HET) c-ros transgenic mouse. The apparent up-regulation in the KO mouse confirmed by Northern and Western blot analyses could be explained by the absence of NaP_i-IIb from the initial segment of the HET epididymis, as revealed by immunohistochemistry, and its presence on the epithelial brush border throughout the proximal epididymis of KO mice, where differentiation of the initial segment fails to occur. Both NaP_i-IIb mRNA and protein were scarce or absent from the cauda epididymidis of both genotypes. A high content of inorganic phosphate was measured enzymatically in the HET cauda luminal fluid, with a 27% decrease in the KO mice. This decrease, presumably from a greater reabsorption of inorganic phosphate, particularly in the initial part of the KO epididymis, may disturb the normal process of sperm maturation in these infertile males. By contrast, no apparent consequences were observed for the transport of Na and Ca² , the concentrations of which (∼26 mM and ∼30 μM, respectively) were measured by microelectrodes to be identical in the caudal fluid from both genotypes.

Germ cell transplantation has tremendous applications in transgenic animal production, assisted reproductive technology, and germline stem cell research. Here, we report for the first time the production of individuals from intraperitoneally transplanted primordial germ cells (PGCs) in animals. To trace the behavior of exogenous PGCs in recipients, PGCs visualized by a green fluorescent protein gene were used as donors. The PGCs prepared from the genital ridges of hatching embryos were transplanted into recipients at various developmental stages. The PGCs injected into the peritoneal cavities of hatching embryos had the ability to migrate toward, and to colonize, the genital ridges of recipient embryos. Furthermore, donor-derived PGCs proliferated and differentiated into mature eggs and sperm in the allogenic gonads; the resulting gametes produced live fry, showing the donor-derived phenotype, through fertilization. Combined with in vitro culture, genetic modification, and cryopreservation of PGCs, this technique provides new approaches for fish bioengineering.

Chronic, low-dose treatment of male rats with cyclophosphamide, a chemotherapeutic agent, is known to affect progeny outcome adversely in a dose-dependent and time-specific manner, resulting in increased pre- and postimplantation loss as well as malformations. Concern exists regarding the genetic quality of mature gametes exposed to cyclophosphamide during mitosis and meiosis. The goal of the present study was to determine the effect of chronic cyclophosphamide treatment during spermatogenesis on the frequency of numerical chromosomal anomalies in epididymal spermatozoa. Male rats were treated with either saline or cyclophosphamide (6 mg kg⁻¹ day⁻¹) for 6 or 9 wk, and cauda epididymal spermatozoa were collected. The rat sperm Y-4 fluorescence in situ hybridization assay was used to assess the induction of spermatozoal disomy, nullisomy, and diploidy involving chromosomes Y and 4. The overall frequency of numerically abnormal spermatozoa was elevated approximately 2-fold (P < 0.001) after 9 wk of cyclophosphamide treatment. Exposure for 9 wk, but not for 6 wk, significantly increased the frequency of spermatozoa with chromosome 4 disomy (P < 0.02) and nullisomy (P < 0.05), but disomy Y and diploidy were not significantly increased with treatment compared to corresponding controls. Independent of treatment, only 27% of aneuploid spermatozoa presented with morphological abnormalities, but all diploid spermatozoa were approximately twice the size of normal cells. Thus, cyclophosphamide disrupts meiotic events before pachynema during spermatogenesis, emphasizing the potential for adverse progeny outcomes following genotoxic damage.

Stress responses are thought to act within the hypothalamopituitary unit to impair the reproductive system, and the sites of action may differ between sexes. The effect of isolation and restraint stress on pituitary responsiveness to GnRH in sheep was investigated, with emphasis on possible sex differences. Experiments were conducted during the breeding season and the nonbreeding season. In both experiments, 125 ng of GnRH was injected i.v. every 2 h into hypothalamopituitary disconnected, gonadectomized rams and ewes on 3 experimental days, with each day divided into two periods. During the second period on Day 2, isolation and restraint stress was imposed for 5.5 h. Plasma concentrations of LH and cortisol were measured in samples of blood collected from the jugular vein. In the second experiment (nonbreeding season), plasma concentrations of epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylglycol were also measured. In both experiments, there was no effect of isolation and restraint stress on plasma concentrations of cortisol in either sex. During the breeding season, there was no effect of isolation and restraint stress on plasma concentrations of LH in either sex. During the nonbreeding season, the amplitude of the first LH pulse after the commencement of stress was significantly reduced (P < 0.05) in rams and ewes. In the second experiment, during stress there was a significant increase (P < 0.05) in plasma concentrations of epinephrine in rams and ewes and significantly higher (P < 0.05) basal concentrations of norepinephrine in ewes than in rams. These results suggest that in sheep stress reduces responsiveness of the pituitary gland to exogenous GnRH during the nonbreeding season but not during the breeding season, possibly because of mediators of the stress response other than those of the hypothalamus-pituitary-adrenal gland axis.

Immune cell trafficking and activity are implicated in the parturition process, but little is known about the role of macrophages in control of uterine contractility at term. In the present study, we tested the hypothesis that endotoxin (lipopolysaccharide [LPS]) enhances uterine contractile activity through a mechanism that involves activation of resident macrophages. Various uterotonins and anti-inflammatory mediators were added to a standard muscle bath preparation that contained strips of uterus from Day 15 pregnant C3H/HeN mice. Spontaneous and agonist-induced contractile activity was enhanced following LPS treatment. LPS increased amplitude but not frequency of contractions. Addition of anti-inflammatory cytokines, interleukin 10 or transforming growth factor β, to suppress macrophage activation did not block LPS-induced increases in contractility. By contrast, indomethacin given to block prostaglandin production suppressed the LPS-induced increase in amplitude of contractions. These findings suggest that an inflammatory response, possibly mediated by activation of macrophages and prostaglandins, participates in the regulation of amplitude but not frequency of contractile activity by the murine uterus before onset of parturition.

Quantitative real-time polymerase chain reaction assay was used to compare the temporal transcriptional activation and mRNA removal for a number of genes in mouse embryos derived by round spermatid injection (ROSI) or intracytoplasmic sperm injection. A number of marker genes with widely different cellular functions were analyzed. Similar patterns of activation were found for the transcription factor Oct 4, the translation initiation factor eukaryotic initiation factor 1A, the L1 ribosomal protein, the chromatin modifying protein histone deacetylase 1, the enzyme hypoxanthine phosphoribosyl transferase, the murine endogenous retrovirus-like element, and the repetitive DNA LINE retrotransposons. Expression of the retrovirus-like mobile element intracisternal A particle, however, was markedly elevated from the two-cell to the blastocyst stages in ROSI embryos. Analyses performed for various paternal mRNAs introduced into the oocyte by the round spermatid, including protamines 1 and 2, transition protein 2, ropporin, and glyceraldehydes 3-phosphate dehydrogenase, revealed all were removed from the preimplantation embryos, albeit with distinct temporal patterns.

A total of 98 898 expressed sequence tags (ESTs) derived from embryos and reproductive tissues in pigs were identified in the GenBank “est_others” database. Pig embryos were collected at 11, 12, 13, 14, 15, 20, 30, and 45 days after gestation. The reproductive tissues were sampled from testis, ovary, endometrium, hypothalamus, anterior pituitary, uterus, and placenta. A gene-oriented approach was developed to annotate these porcine EST sequences to census the genes expressed from these sources. Of the 33 308 mRNA sequences from the human genes used as references (data accessed on 1 November 2002), 9410 had the porcine EST homologs expressed in embryos and 11 795 had the EST homologs expressed in reproductive tissues. The entire genome contributes at least 28.3% of its genes to embryo development and 35.4% of its genes to reproduction. Using the EST entry numbers as indicators of gene expression, we determined that the gene expression patterns differ significantly between embryos and reproductive tissues in pigs. The basic active genes were identified for each source, but most of them are not coexpressed abundantly. Few genes were expressed on the Y chromosome (P < 0.01), but they may represent counterparts of the double-dose genes that remain active in an inactivated X chromosome in females but are needed for proper development and growth. The census provides a panel of transcripts in a broad sense that can be used as targets to study the mechanisms involved in embryo development and reproduction in pigs and other mammals, including humans.

Arylsulfatase A (AS-A) is localized to the sperm surface and participates in sperm-zona pellucida binding. We investigated how AS-A, usually known as an acrosomal enzyme, trafficked to the sperm surface. Immunocytochemistry of the mouse testis confirmed the existence of AS-A in the acrosomal region of round and elongating spermatids. However, immunofluorescence and flow cytometry indicated the absence of AS-A on the surface of live testicular sperm. In contrast, positive AS-A staining was observed in the heads of live caudal epididymal and vas deferens sperm. The results suggested that acquisition of AS-A on the sperm surface occurred during epididymal transit. Immunocytochemistry of the epididymis revealed AS-A in narrow and apical cells in the initial segment and in clear cells in all epididymal regions. However, these epithelial cells are in the minority and are not involved in secretory activity. In the caudal epididymis and vas deferens, AS-A was also localized to principal cells, the major epithelial cells. Because principal cells have secretory activity, they may secrete AS-A into the epididymal fluid. This hypothesis was supported by our results revealing the presence of AS-A in the epididymal and vas deferens fluid (determined by immunoblotting and ELISA) and an AS-A transcript in the epididymis (by reverse transcription polymerase chain reaction). Alexa-430 AS-A bound to epididymal sperm with high affinity (K_d = 46 nM). This binding was inhibited by treatment of sperm with an antibody against sperm surface sulfogalactosylglycerolipid. This finding suggests that AS-A in the epididymal fluid may deposit onto sperm via its affinity to sulfogalactosylglycerolipid.

The postimplantation developmental potential of embryos can be affected by various forms of cell death, such as apoptosis, at preimplantation stages. However, correct assessment of apoptosis is needed for adequate inference of the developmental significance of this process. This study is the first to investigate the independent chronological occurrence of apoptotic changes in nuclear morphology and DNA degradation (detected by the TUNEL reaction) and incidences of nuclei displaying these features at various preimplantation stages of bovine embryos produced both in vivo and in vitro. Different elements of apoptosis were observed at various developmental stages and appeared to be differentially affected by in vitro production. Nuclear condensation was observed from the 6-cell stage in vitro and the 8-cell stage in vivo, whereas the TUNEL reaction was first observed at the 6-cell stage in vitro and the 21-cell stage in vivo. Morphological signs of other forms of cell death were also observed in normally developing embryos produced both in vivo and in vitro. The onset of apoptosis seems to be developmentally regulated in a stage-specific manner, but discrete features of the apoptotic process may be differentially regulated and independently modulated by the mode of embryo production. Significant differences in indices of various apoptotic features were not evident between in vivo- and in vitro-produced embryos at the morula stage, but such differences could be observed at the blastocyst stage, where in vitro production was associated with a higher degree of apoptosis in the inner cell mass.

Testicular secretion of estradiol is necessary for normal spermatogenesis and male reproductive physiology in humans and rodents. The role of estradiol in nonmammalian vertebrates remains unknown, but elevated circulating estradiol has been reported in male lizards, alligators, and various bird species. We have been unable to detect circulating estradiol in male alligators; therefore, we reexamined the question of testicular production of estradiol in alligators using more rigorous assay procedures. A large pool of plasma from a male alligator was extracted and run through an HPLC column. Immunoreactive estradiol-like material eluted coincident with authentic estradiol. By using an ultrasensitive RIA and processing large volumes of male plasma (1000 μl), we were able to measure estradiol. Estradiol in male alligators ranged from 0.23 to 3.14 pg/ml, whereas estradiol in immature female alligators ranged from 14 to 66 pg/ml. Aromatase activity in microsomes from adult alligator ovarian tissue was 36.2 ± 1.6 pmol mg⁻¹ h⁻¹, whereas activity in testicular microsomes ranged between 0.92 and 2.38 pmol mg⁻¹ h⁻¹. Ovarian aromatase activity was inhibited in a concentration-dependent fashion by Fadrozole, but the essentially background activity of testicular aromatase was not inhibited at any concentration of Fadrozole. Likewise, a comparison of alligator testicular and ovarian aromatase mRNA expression gave a similar result: the ovarian expression was 600-fold higher and brain tissue was 10-fold higher than that of the testis. Circulating estradiol in male alligators is probably of extragonadal origin, and the testis produces little if any of this steroid.

Several researchers have suggested recently that the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst is orthogonal to the first cleavage plane of the two-cell embryo. To determine the universality of this relationship, we used embryos of two different genotypes, F₁ (C57BL/6 × DBA/2) and CD-1. The position of the first cleavage plane in the early blastocyst was determined by labeling a blastomere with the fluorescent lineage tracer DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) at the two-cell stage. Approximately one quarter of the blastocysts from both genotypes possessed an Em-Ab axis that respected the orthogonal relationship with the first cleavage plane. However, the remainder of the blastocysts deviated from the orthogonal relationship. This result indicates that the orthogonal orientation of the Em-Ab axis to the first cleavage plane is not a universal phenomenon. We also tested whether the angular relationship between the Em-Ab axis and first cleavage plane influences postimplantation embryo development. We sorted the blastocysts that had the Em-Ab axis orthogonal to the first cleavage plane from the ones that did not. These two types of blastocysts were transferred separately into surrogates, and fetal development was examined in late gestation. The results revealed that both types of blastocysts produced normal fetuses at a similar frequency. Thus, the relationship of the blastocyst axis to the first cleavage plane does not significantly influence later development.

Five early-treated and four late-treated prenatally androgenized and five normal female rhesus monkeys were studied to determine whether prenatal testosterone propionate exposure beginning Gestational Days 40–44 (early-treated) or 100–115 (late-treated) affects follicular steroidogenesis during recombinant human FSH (rhFSH) treatment. All monkeys underwent rhFSH injections, without human chorionic gonadotropin administration, followed by oocyte retrieval. Serum FSH, LH, estradiol (E₂), progesterone (P), 17α-hydroxyprogesterone (17 OHP), androstenedione (A₄), testosterone, and dihydrotestosterone were measured basally during rhFSH therapy and at oocyte retrieval. Follicle fluid (FF) sex steroids, oocyte fertilization, and embryo development were analyzed. Circulating FSH, E₂, 17 OHP, A₄, and dihydrotestosterone levels increased similarly in all females. Serum LH levels decreased from basal levels in normal and late-treated prenatally androgenized females but were unchanged in early-treated prenatally androgenized females. Serum P levels at oocyte retrieval were comparable with those before FSH treatment in all females. All prenatally androgenized females showed reduced FF levels of A₄ and E₂ but not P or dihydrotestosterone. Intrafollicular T concentrations also were significantly lower in late-treated compared with early-treated prenatally androgenized females or normal females. In early-treated prenatally androgenized females, but not the other female groups, intrafollicular A₄ and E₂ levels were reduced in follicles containing oocytes that failed fertilization or produced zygotes with cleavage arrest before or at the five- to eight-cell embryo stage. Therefore, in monkeys receiving rhFSH therapy alone without human chorionic gonadotropin administration, early prenatal androgenization reduced FF concentrations of E₂ and A₄ in association with abnormal oocyte development, without having an effect on P, testosterone, or dihydrotestosterone concentrations.

AP-2γ is a member of the AP-2 transcription factor family, is highly enriched in the trophoblast cell lineage, and is essential for placental development. In an effort to identify factors regulating AP-2γ gene expression, we isolated and characterized the promoter and 5′-flanking region of the mouse and human AP-2γ genes. The transcription start site of the mouse AP-2γ gene was mapped by primer extension and 5′ rapid amplification of cDNA ends. Deletion analysis of the 5′-flanking region revealed a 704-base pair (bp) sequence located approximately 6 kilobases (kb) upstream of the transcription start site that is required for enhanced expression in trophoblast cells. Additional gene transfer studies showed that basal promoter activity resides within a highly conserved, approximately 200-bp DNA sequence located immediately upstream of the transcription start site. The conserved region is highly GC-rich and lacks typical TATA or CCAAT boxes. Multiple potential Sp- and AP-2-binding sites are clustered within this region. Electrophoretic mobility shift assays demonstrated that Sp1 and Sp3 bind to three sites in the promoter region of the mouse AP-2γ gene. Combined mutation of the three putative Sp sites reduced promoter activity by 80% in trophoblast and nontrophoblast cells, demonstrating the functional importance of these sites in regulating AP-2γ gene expression. In summary, we have identified a potential trophoblast cell-specific regulatory element located approximately 6 kb upstream of the murine AP-2γ gene transcription start site, and we have shown that Sp1 and Sp3 bind to cis-regulatory elements located in the promoter proximal region and contribute to basal promoter activity.

The testicular vasculature is unique in several ways. The unfenestrated endothelial cells constitute one part of the blood-testis barrier, and testicular microvessels are normally resistant to inflammation mediators. At the same time that angiogenic factors and inflammation mediators are constitutively produced, the proportion of proliferating endothelial cells is considerably higher than in other organs, but new blood vessels are not formed. Hormonal stimulation of the testis with hCG increase endothelial cell proliferation, vascular permeability, and sensitivity to locally injected inflammation mediators. In the present study, we examined whether local expression of angiopoietin (ang) 1, an inhibitor of vascular leakage and sprouting angiogenesis, and its antagonist, ang 2, could be involved in establishing this vascular phenotype. Using reverse transcription-polymerase chain reaction and immunohistochemistry, we demonstrate that testicular vascular endothelial growth factor-A (VEGF-A), ang 1, ang 2, and the ang-receptor tie 2 are expressed in the testis and that hormonal stimulation with hCG is accompanied by increased expression of VEGF-A and ang 2. The ang 1 protein is expressed in testicular microvessels under basal conditions, and it is largely unaffected after hCG stimulation. Expression of ang 2 in microvessels, in contrast, is low under basal conditions and is up-regulated by hCG. Intratesticular injection of human recombinant ang 1 protein inhibits hCG-induced increase in vascular permeability. Injection of ang 2 in the testis increases endothelial cell proliferation and the volume of the interstitial space. We therefore suggest that ang 1 stabilizes testicular microvessels under basal conditions and that a shift in this balance caused by increased ang 2, together with increased VEGF-A, allows vascular leakage, high endothelial cell proliferation, and presumably, vascular growth after hormonal stimulation.

The present study was undertaken to identify the mechanisms underlying the effect of transforming growth factor (TGF) β on FSH receptor (FSH-R) in rat granulosa cells. Compared to the control, the treatment of granulosa cells with TGFβ (10 ng/ml) increased FSH-R mRNA transcripts (5.5 and 2.4 kilobases) in a time-dependent manner, with a maximum increase of approximately 2-fold at 48 h. We then investigated whether the effect of TGFβ on FSH-R mRNA levels was the result of increased transcription and/or altered mRNA stability. To determine whether the FSH-R 5′-flanking region plays a role in directing FSH-R mRNA expression, the proximal area of the FSH-R 5′-flanking regions were inserted into an expression vector, pGL-Basic, which contains luciferase as the receptor gene, and the resulting plasmids were transiently transfected into rat granulosa cells. The FSH (30 ng/ml) significantly enhanced the activity of 1862 base pairs of the FSH-R 5′-flanking region, but treatment with TGFβ did not significantly influence the activity induced by FSH. On the other hand, the decay curves for FSH-R mRNA transcript in primary granulosa cells showed a significant increase in half-life after the addition of TGFβ. Transforming growth factor β stimulates the expression of follistatin mRNA accumulation in a dose- and time-dependent manner. Treatment with activin produced a substantial increase in FSH-R mRNA level. Concurrent treatment with follistatin neutralized this activin effect on FSH-R mRNA, as reported, although concurrent treatment with follistatin did not affect TGFβ-induced FSH-R mRNA. Therefore, the profile of the TGFβ effect on FSH-R mRNA granulosa cells may be caused by the increased stability of FSH-R mRNA and insensitivity to the follistatin.

In an attempt to find a suitable freezing method for goat semen, two experiments were conducted to study the influence of trehalose on the cryopreservation of goat spermatozoa. In experiment 1, goat spermatozoa were frozen in trehalose extender (0.375 M) alone (100%) or at different combinations of trehalose with Tris-citric acid-glucose (TCG) extender (0%, 25%, 50%, 75%). Final concentrations of 20% (v:v) egg yolk and 4% (v:v) glycerol were employed in the extenders (osmolality = 370, pH = 7). Sperm motility was assessed using a computer-assisted sperm analysis system and acrosome integrity was assessed using fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA). The sperm-motility parameters improved significantly by increasing the concentration of trehalose (P < 0.05) and significantly high recovery rates for the motility parameters were also achieved by a high concentration of trehalose (P < 0.05). Motility of the frozen-thawed spermatozoa after a 3-h incubation improved significantly with increasing concentrations of trehalose in the extender (P < 0.05). The 75% and 100% trehalose extenders yielded a significant increase in the percentage of spermatozoa with intact acrosome (P < 0.05). In experiment 2, merocyanine 540/Yo-Pro staining was used to study the influence of trehalose on membrane fluidity compared with that of sucrose and TCG. Percentage of cells with high merocyanine fluorescence was significantly higher in spermatozoa treated with trehalose than sucrose or TCG (P < 0.05), indicating a significantly highest membrane fluidity of sperm samples extended with trehalose solution. We thus conclude that the substitution of a Tris-citric acid diluent composition with trehalose significantly improves the freezability of goat spermatozoa. Furthermore, the cryoprotective effects of trehalose observed in this study may be due to enhanced sperm membrane fluidity before freezing.

Conceptus-uterine communication is established during trophoblastic elongation when the conceptus synthesizes and releases estrogen, the maternal recognition signal in the pig. Interleukin-1β (IL-1β) is a differentially expressed gene during rapid trophoblastic elongation in the pig. The current investigation determined conceptus and endometrial changes in gene expression for IL-1β, IL-1 receptor antagonist (IL-1Rant), IL-1 receptor type 1 (IL-1RT1), and IL-1 receptor accessory protein (IL-1RAP) in developing peri- and postimplantation conceptuses as well as uterine endometrium collected from cyclic and pregnant gilts. Conceptus IL-1β gene expression was enhanced during the period of rapid trophoblastic elongation compared with earlier spherical conceptuses, followed by a dramatic decrease in elongated Day 15 conceptuses. IL-1RT1 and IL-1RAP gene expression was greater in Day 12 and 15 filamentous conceptuses compared with earlier morphologies while IL-1Rant gene expression was unchanged by conceptus development. The uterine lumenal content of IL-1β increased during the process of trophoblastic elongation on Day 12. Uterine IL-1β content declined on Day 15, reaching a nadir by Day 18 of pregnancy. IL-1β gene expression in porcine conceptuses was temporally associated with an increase in endometrial IL-1RT1 and IL-1RAP gene expression in pregnant gilts. Endometrial IL-1β and IL-1Rant gene expression were lowest during Days 10–15 of the estrous cycle and pregnancy. The temporal expression of IL-1β during conceptus development and the initiation of conceptus-uterine communication suggests conceptus IL-1β synthesis plays an important role in porcine conceptus elongation and the establishment of pregnancy in the pig.

Transplantation of spermatogonial stem cells into syngeneic or immunosuppressed recipient mice or rats can result in donor-derived spermatogenesis and fertility. Recently, this approach has been employed to introduce a transgene into the male germline. Germ-cell transplantation in species other than laboratory rodents, if successful, holds great promise as an alternative to the inefficient methods currently available to generate transgenic farm animals that can produce therapeutic proteins in their milk or provide organs for transplantation to humans. To explore whether germ-cell transplantation could result in donor-derived spermatogenesis and fertility in immunocompetent recipient goats, testis cells were transplanted from transgenic donor goats carrying a human alpha-1 antitrypsin expression construct to the testes of sexually immature wild-type recipient goats. After puberty, sperm carrying the donor-derived transgene were detected in the ejaculates of two out of five recipients. Mating of one recipient resulted in 15 offspring, one of which was transgenic for the donor-derived transgene. This is the first report of donor cell-derived sperm production and transmission of the donor haplotype to the next generation after germ-cell transplantation in a nonrodent species. Furthermore, these results indicate that successful germ-cell transplantation is feasible between immunocompetent, unrelated animals. In the future, transplantation of genetically modified germ cells may provide a more efficient alternative for production of transgenic domestic animals.

The growth and development of follicles within the ovary are highly dependent on autocrine and paracrine signaling involving growth factors from granulosa cells, theca cells, stromal interstitial cells, and the oocytes. The growth factor bone morphogenetic protein-4 (BMP-4) and its receptor (BMPR-IB) have been detected in ovaries, and a mutation in BMPR-IB has been associated with abnormal ovulation rate. The objective of the current study was to examine the role that BMP-4 plays in the early stages of primordial follicle development. Ovaries from 4-day-old rats were placed into a whole-ovary organ culture system for 2 wk to investigate the effect that treatment with exogenous BMP-4 has on early follicle development. BMP-4-treated ovaries had a significantly higher proportion of developing primary follicles and fewer arrested primordial follicles than did untreated controls. This indicates that BMP-4 promotes primordial follicle development and the primordial-to-primary follicle transition. Ovaries were also treated with neutralizing antibody against BMP-4 to determine effects of removing endogenously produced BMP-4. Interestingly, ovaries treated with BMP-4 antibody were markedly smaller than controls. This was associated with a progressive loss of oocytes and primordial follicles, a progressive increase in cellular apoptosis, and an accompanying loss of normal ovarian tissue morphology over time. Immunocytochemistry localized BMP-4 protein to isolated stromal cell populations, selected stromal cells (i.e., pretheca cells) associated with developing primordial follicles, and the basement membrane of follicles. Ovaries were treated with BMP-4 and RNA collected after organ culture to determine whether BMP-4 signaling affects expression of other growth factors. Kit ligand and basic fibroblast growth factor expression was unchanged, but TGFα expression was decreased in whole ovaries. Taken together, these data suggest that BMP-4 plays an important role in promoting the survival and development of primordial follicles in the neonatal ovary.

Menstruation and endometrial regeneration occur during every normal reproductive cycle in women and some Old World primates. Many of the cellular and molecular events of menstruation have been identified by correlative or in vitro studies, but the lack of a convenient model for menstruation in a laboratory animal has restricted functional studies. In this study, a mouse model for menstruation first described by Finn in the 1980s has been modified for use in a commonly used inbred strain of mouse. A decidual stimulus was applied into the uterine lumen of appropriately primed mice and leukocyte numbers and apoptosis were examined over time following progesterone withdrawal. Endometrial tissue breakdown was initiated after 12–16 h, and by 24 h, the entire decidual zone had been shed. Re-epithelialization was nearly complete by 36 h and the endometrium was fully restored by 48 h. Leukocyte numbers increased significantly in the basal zone by 12 h after progesterone withdrawal, preceding stromal destruction. Stromal apoptosis was detected by TUNEL staining at 0 and 12 h but decreased by 16 h after progesterone withdrawal. This mouse model thus mimics many of the events of human menstruation and has the potential to assist in elucidation of the functional roles of a variety of factors thought to be important in both menstruation and endometrial repair.

Adult Follitropin Receptor Knockout (FORKO) female mice are infertile and estrogen deficient. In order to understand the peri/postnatal developmental changes, we have now characterized the structural and molecular aberrations by comparing several markers of follicular development in 2-, 10-, and 24-day-old wild-type and FORKO females. By Day 24, FORKO mice have 40%–50% smaller uteri and vaginas. Estradiol is undetectable but testosterone and LH levels are already elevated at this age. FORKO ovaries are 45% smaller, indicating a postnatal or perinatal deficit consequent to FSH receptor ablation. This is attributable to decreased numbers of growing follicles and reduced diameter. Developmental markers, such as Müllerian inhibiting substance, GATA-4, estrogen receptor β, and androgen receptor, were differentially expressed in granulosa cells. In the 2-day-old mutant neonates, a faster recruitment process was noted that later slowed down, impeding development of follicles. This is noteworthy in light of the controversy regarding the direct role of FSH/receptor system as a determinant of small and preantral follicle development in rodents. As the pool of nongrowing primordial follicles specifies the duration of female fertility and timing of reproductive senescence, we believe that the postnatal FORKO female mouse could help in exploring the signals that impact on early folliculogenesis. In addition, our data suggest that the FSH/receptor system is a major contributor to the formation and recruitment of the nongrowing pool of follicles as early as Postnatal Day 2 in the mouse.

Targeted disruption of the mouse FSH receptor gene (FSH-R) that mediates the action of the FSH results in a gene dose-related ovarian phenotype in the developing as well as the adult animal. While null females (FORKO) are sterile, the haplo-insufficient mice experience early reproductive senescence. The purpose of this study was to first record changes in oocyte development in the null FORKO and haplo-insufficient mice. Oocyte growth is significantly retarded in the null mutants with thinner zona pellucida in preantral follicles, but thicker zona pellucida in secondary follicles. This morphometric change indicates developmental aberrations in coordination of the germ cell (oocyte) and the somatic granulosa cell (GC) compartments. Markers for primordial germ cell proliferation and oocyte growth, such as the c-Kit/Kit-ligand and bone morphogenetic protein-15 (BMP-15) were downregulated in both null and /− ovaries, suggesting disrupted communication between oocyte and GCs. Extensive changes in the expression of other oocyte-specific gene products like the zona pellucida glycoproteins (zona pellucida A, B, and C) indicate major alteration in the extracellular matrix surrounding the germ cells. This led to leaky germ cells that allowed infiltration of somatic cells. These results show that the loss of FSH-R signaling alters the follicular environment, where oocyte-granulosa interactions are perturbed, creating an out-of-phase germ cell and somatic cell development. We believe that these data provide an experimental paradigm to explore the mechanisms responsible for preserving the structural integrity and quality of oocytes at different ages.

Although the mammalian germinal stem cell (GSC) provides a good model to investigate the regulation of stem cells, the small number of these cells currently available hampers elucidation of the regulatory mechanism. Here, we show the dramatic amplification of GSCs in mouse testis following transfection of human glial cell line-derived neurotrophic factor cDNA into Sertoli cells using an efficient, in vivo electroporation technique. Transplantation analysis demonstrated not only GSC enrichment but also differentiation from stem cells into sperm. The GSC population, as estimated using a colony-formation assay, was approximately 20-fold greater than in cryptorchid testis, or approximately 500- to 1000-fold greater than in normal adult testis. This system should provide sufficient quantities of GSCs to accelerate our understanding of GSC properties, regulation mechanisms, and behavior control.

The development and functions of female reproductive tissues are regulated by the actions of two major sex steroid hormones, estrogen and progesterone. To investigate estrogen-dependent gene expression in the rat uterus, we studied the effect of ovariectomy with or without estrogen treatment on the uterine expression of 3000 genes using cDNA microarrays. Many genes were regulated by either treatment, but only few were reciprocally regulated by these contrasting treatments. The present study confirms previous findings and identifies several genes with expressions not previously known to be influenced by estrogen. These genes include follistatin-related protein, Thy-1 glycoprotein, α-fodrin, CD24, immediate early response 5, insulin-like growth factor-binding protein 2, growth response protein CL-6 (INSIG-1), ladinin1, class I major histocompatibility complex heavy chain, lactadherin, ezrin, and Fas-activated serine/threonine kinase. Because of their function as regulators of proliferation and apoptosis, CD24, insulin-like growth factor-binding protein 2, and Fas/Fas ligand were examined further by immunohistochemical expression and tissue localization analysis. Our analysis confirms a contrasting regulation of these gene products by ovariectomy and estrogen treatment. The present study identifies novel mediators of estrogen actions in the uterus and provides genome-wide expression data from which novel hypotheses regarding uterine function can be generated.

Pituitary gland growth hormone (GH) secretion is influenced by two hypothalamic neuropeptides: growth hormone-releasing hormone (GHRH) and somatostatin. Recent data also suggest that estrogen modulates GH release, particularly at the time of the preovulatory luteinizing hormone surge, when a coincident surge of GH is observed in sheep. The GHRH neurons do not possess estrogen receptor α (ERα), suggesting that estrogen does not act directly on GHRH neurons. Similarly, few somatotropes express ERα, suggesting a weak pituitary effect of estradiol on GH. It was hypothesized, therefore, that estradiol may affect somatostatin neurons to modulate GH release from the pituitary. Using immunocytochemical approaches, the present study revealed that although somatostatin neurons were located in several hypothalamic sites, only those in the arcuate nucleus (13% ± 2%) and ventromedial nucleus (VMN; 29% ± 1%) expressed ERα. In addition, we found that all neurons immunoreactive for somatostatin-14 were also immunoreactive for somatostatin-28(1–12). To determine whether increased GH secretion in response to estradiol is through modulation of GHRH and/or somatostatin neuronal activity, a final study investigated whether c-fos expression increased in somatostatin- and GHRH-immunoreactive cells at the time of the estradiol-induced LH surge in intact anestrous ewes. Estradiol significantly (P < 0.05) increased the percentage of GHRH (estradiol, 75% ± 3%; no estradiol, 19% ± 2%) neurons expressing c-fos in the hypothalamus. The percentage of somatostatin-immunoreactive neurons coexpressing c-fos in the estradiol-treated animals was significantly (P < 0.05) higher (periventricular, 44% ± 3%; arcuate, 72% ± 5%; VMN, 81% ± 5%) than in the control animals (periventricular, 22% ± 1%; arcuate, 29% ± 3%; VMN, 31% ± 3%). The present study suggests that estradiol modulates the activity of GHRH and somatostatin neurons but that this effect is most likely mediated through an indirect interneuronal pathway.

To study the complex molecular mechanisms of mammalian spermatogenesis, it would be useful to be able to isolate cells at each stage of differentiation, especially at the stage in which the cells switch from mitosis to meiosis. Currently, no useful marker proteins or gene promoters specific to this important stage are known. We report here a transgenic mouse line that under the control of the promoter for a histone variant, H2A.X, expressed an enhanced green fluorescent protein (EGFP) in cells at the stage of the mitosis-meiosis switch. Endogenous H2A.X is expressed in type A spermatogonia through meiotic prophase spermatocytes in testis and in some somatic cells. However, despite the fact that its expression was driven by the H2A.X promoter, the EGFP expressed in the transgenic mice specifically labeled only the intermediate spermatogonia stage through the meiotic prophase spermatocyte stage in transgenic mice containing the −600-base pair H2A.X promoter/EGFP construct. Type A spermatogonia and somatic cells of other organs were not labeled. This expression pattern made it possible to isolate living cells from the testis of the transgenic mice at the stage of the mitosis-meiosis switch in spermatogenesis using EGFP fluorescence.

The members of the nectin/CD155 gene family represent a growing class of novel cell adhesion molecules of the immunoglobulin superfamily. In the present study, we describe the generation of a mouse line lacking a functional nectin-2 gene (nectin-2^LacZ/LacZ) and analyze the resulting male-specific infertility phenotype. Although nectin-2^LacZ/LacZ males produced normal amounts of motile spermatozoa, scanning electron microscopy revealed severe malformations of the spermatozoan head and midpiece. Besides a 4-fold reduction in migration of nectin-2^LacZ/LacZ spermatozoa to the oviducts, in vitro binding to zona-intact mouse oocytes was reduced 6-fold. On the other hand, nectin-2^LacZ/LacZ spermatozoa bound to zona-free hamster oocytes at near-wild type levels but, remarkably, failed to penetrate. In addition to the previously reported expression of nectin-2 and nectin-3 at Sertoli-spermatid junctions and of nectin-2 at inter-Sertoli cell junctions, we also found nectin-2 to localize at apical cell-cell junctions of the epididymal epithelium. Expression analysis of a LacZ knockin gene into the defunct nectin-2 gene in nectin-2^LacZ/LacZ mice provided additional support for our earlier conjecture that in normal testis, nectin-2 is produced exclusively by Sertoli cells. Finally, we found Sertoli-spermatid junctions in nectin-2^LacZ/LacZ mice to be virtually devoid of the actin-bundling protein espin, suggesting that ectoplasmic specializations fail to form in the absence of nectin-2. Our functional analyses indicate that the infertility phenotype of nectin-2-deficient male mice is caused by a combination of reduced migration to the oviduct, spermatozoa-zona binding, and sperm-oocyte fusion. We corroborate our previous description of a heterotypic adhesion complex between Sertoli cells and elongated spermatids that is maintained by nectin-2 and nectin-3, respectively.

Tumor necrosis factor (TNF) α is an important physiological mediator of cell-to-cell communication. Recent observations suggest that TNFα is involved in the control of reproductive functions. The present study examined the role of TNFα in the secretion of factors involved in regulating smooth muscle contraction, such as prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α), endothelin-1 (ET-1), and angiotensin II (Ang II), as it was in the original by the cow oviduct at different stages of the estrous cycle using an in vitro microdialysis system. Expression of mRNA for TNFα and its receptors (TNFα-R) was also evaluated. For microdialysis, the lumen of a portion (length, 10 cm) of the each oviductal segment was implanted with a dialysis capillary membrane, and TNFα (100 ng/ml) was infused for 4–8 h during a 16-h incubation period. The microdialysis system maintains cell-to-cell integrity and cell-to-cell communication, and it enables real-time observation of physiological changes in the luminal release of different substances. Concentrations of PG, ET-1, and Ang II in 4-h fractions were measured using second-antibody enzyme immunoassays. Infusion of TNFα stimulated oviductal secretion of PG, ET-1, and Ang II during the follicular and postovulatory stages, but not during the luteal stage. Expression of TNFα, TNFα-R type I, and TNFα-R type II mRNA was detected in the bovine oviduct by reverse transcription-polymerase chain reaction. High expression of both TNFαR types and ligands was detected during the follicular and postovulatory stages, whereas low expression was detected during the luteal stage. The results of the present study provide, to our knowledge, the first direct evidence that TNFα stimulates PG, ET-1, and Ang II secretion and that up-regulation of the TNFα system occurs in the cow oviduct during the periovulatory period. In conclusion, the TNFα system may optimize the release of contraction-related substances and modulate local contraction to regulate the oviductal transport of the gametes and embryo.

The relationship between abnormal sperm morphology and chromosomal aberrations has been of interest. Thus far, however, studies have focused on frequencies of sperm with either abnormal morphology or aneuploidies in semen samples, not on detection of individual spermatozoa exhibiting both abnormal morphology and aneuploidy. To assess the feasibility of simultaneous evaluation of both attributes in an individual sperm cell, we investigated whether sperm shape is preserved after decondensation and denaturation, procedures that are required for fluorescent in situ hybridization (FISH). On 21 slides, 395 sperm were fixed, photographed, and then digitized by the computer-assisted Metamorph morphometry program for individual evaluation before decondensation. To establish whether sperm of various shapes would behave in similar manners, the cells were also classified, according to their head shapes, into symmetrical (n = 115), asymmetrical (n = 115), irregular (n = 115), and amorphous (n = 50) categories. Following decondensation and subsequent denaturation, sperm that had been photographed initially were relocalized and digitized for morphometry. Head area, perimeter, long axis, short axis, shape factor, and tail length were evaluated in each of the 395 sperm in both the native and decondensed states. After the decondensation and denaturation protocol of the FISH procedure, the sperm exhibited a proportional increase in dimensions as compared to their original sizes. Their initial shapes were preserved with high fidelity whether the sperm were in the symmetrical, asymmetrical, irregular, or amorphous categories. Hybridization with the chromosome probes had no further effect on sperm shape or size. We provide images to demonstrate how these findings facilitate studies about the relationship between sperm shape and chromosomal content or aberrations in individual spermatozoa.

Mammalian gonadotropin-releasing hormone (GnRH) I is the neuropeptide that regulates reproduction. In recent years, a second isoform of GnRH, GnRH II, and its highly selective type II GnRH receptor were cloned and identified in monkey brain, but its physiological function remains unknown. We sought to determine whether GnRH II stimulates LH and FSH secretion by activating specific receptors in primary pituitary cultures from male monkeys. Dispersed pituitary cells were maintained in steroid-depleted media and stimulated with GnRH I and/or GnRH II for 6 h. Cells were also treated with Antide (Bachem, King of Prussia, PA), a GnRH I antagonist, to block gonadotropin secretion. In monkey as well as rat pituitary cultures, GnRH II was a less effective stimulator of LH and FSH secretion than was GnRH I. In both cell preparations, Antide completely blocked LH and FSH release provoked by GnRH II as well as GnRH I. Furthermore, the combination of GnRH I and GnRH II was no more effective than either agonist alone. These results indicate that GnRH II stimulates FSH and LH secretion, but they also imply that this action occurs through the GnRH I receptor. The GnRH II receptors may have a unique function in the monkey brain and pituitary other than regulation of gonadotropin secretion.

The nuclei of guinea pig spermatogonia and spermatocytes were studied by means of quantitative autoradiography and electron microscopic methods such as high-resolution cytochemistry, immunocytochemistry, and in situ hybridization. Our observations reveal, in the nucleus of spermatogonia type B, small lampbrush structures of extended chromatin not found in nonmeiotic cells. During meiotic interphase, pairs of parallel lampbrush structures become associated by numerous filaments. The formation of the synaptonemal complex is simultaneous with the extension of chromosomal axes in a continuous leptotene-zygotene stage. Some chromosomes do not recognize their homologs before the onset of the leptotene-zygotene stage and undergo classical leptotene and zygotene stages. The immunocytochemical localization of Dmc1 and Rad51 supports the idea that these proteins are not involved in homology search and final pairing. Immunolocalization of DNA, RNA polymerase II, heterogeneous nuclear ribonucleoproteins, small nuclear ribonucleoproteins, and the trimethyl-guanosin cap of small nuclear RNAs suggests that the chromatin of lampbrush structures transcribe hnRNA and that splicing is scarce. The results of quantitative autoradiography after [³H]uridine labeling show an intense transcription accompanied by a very slow export of RNA. In situ hybridization demonstrates the presence of RNA in the regions of homology recognition and pairing. These results lead us to propose that the RNA synthesized in the lampbrush structures is involved in the process of homology searching and recognition.

Bovine embryos produced in vitro differ considerably in quality from embryos developed in vivo. The in vitro production system profoundly affects the competence to form blastocysts, the number of cells of the total embryo and of the inner cell mass (ICM), and the incidence of apoptosis. To our knowledge, the effects of different postfertilization regimens before and after completion of the fourth embryonic cell cycle on these aspects have not yet been investigated. In the present study, we assessed the blastulation rate by stereomicroscopy and the cell number of the total embryo, of the ICM, and of the cells with apoptotic changes by confocal laser-scanning microscopy after staining with propidium iodide and TUNEL. Two groups of embryos were developed in heifers, after superovulation, until 45 or 100 h postovulation (po) and, after collection on slaughter, were further cultured in vitro until Day 7 po. A third and fourth group comprised embryos that were produced entirely in vitro or in vivo. The results indicate that passage in vivo of the fourth cell cycle does not prevent acceleration of the formation of the blastocoele in vitro but may be the critical factor contributing to a higher cell number in the total blastocyst and its ICM. The lower quality of in vitro-produced embryos can be attributed to the ICM having less viable cells because of a lower number of cells and a higher incidence of apoptosis that appears to be determined before completion of the fourth cell cycle.

During reproductive life, only a selected few ovarian follicles mature and ovulate, while the vast majority of follicles undergo a degenerative process called atresia. Recent studies have indicated that follicular atresia is mediated through apoptosis of follicular granulosa cells. The objectives of the present study were to determine the time of onset of apoptosis in granulosa cells of preovulatory follicles and to evaluate the consequences of gonadotropin withdrawal on mitogen-activated protein (MAP) kinase activities. Bonnet monkeys (Macaca radiata) undergoing controlled ovarian stimulation cycles were utilized for stimulation of multiple follicles, and granulosa cells were retrieved from preovulatory follicles at 24, 48, 72, and 96 h after stopping gonadotropin treatment. Serum and follicular fluid estradiol concentrations were highest at 24 h but declined precipitously (P < 0.05) to reach the lowest concentrations at 96 h; however, progesterone concentrations during this period did not increase, indicating the absence of luteinization. Quantitative analysis of genomic DNA by 3′-end labeling revealed the presence of low-molecular-weight fragments from 48 h onward, but by agarose gel electrophoresis, DNA laddering could be visualized only after 72 h. Messenger RNA expression for Bax, caspase-2, and caspase-3 increased with the onset of apoptosis. Immunoblot analysis of MAP kinases in lysates of granulosa cells (48–72 h) indicated increased (P < 0.05) levels of phosphorylated extracellular response kinase-1 and -2, Jun N-terminal kinase (JNK)-1 and -2, and p38. However, in vitro kinase assay data indicated that only phospho-JNK and -p38 activities were higher at 72 h compared to 24 h. These results demonstrate that granulosa cells of preovulatory follicles undergo apoptosis and that increased activities of phospho-JNK and -p38 are correlated with apoptosis in the primate.

Perifollicular angiogenesis is closely associated with ovarian follicular development. To investigate whether additional induction of perifollicular angiogenesis would support subsequent follicular development, we directly injected vascular endothelial growth factor (VEGF) gene fragments into the ovaries of miniature gilts, followed by gonadotroph treatment to stimulate follicle growth. In addition, to confirm extraexpression of the VEGF gene after injection, we assessed the expression of two isoforms of VEGF (VEGF 120 and VEGF 164) in granulosa cells and expression of fms-like tyrosine kinase (Flt-1), expression of fetal liver kinase (Flk-1), and density of capillary networks in theca cells. Direct injection of VEGF gene fragments into the ovaries was performed 7 days before eCG treatment. The ovaries in miniature gilts were removed 72 h after eCG treatment for histological examination. Granulosa cells and thecal tissues in the antral follicles (diameter, >4 mm) were collected to detect the mRNA expression of VEGF isoforms in the granulosa cells and of Flt-1 and Flk-1 in the thecal tissues by semiquantitative reverse transcription-polymerase chain reaction. The VEGF levels were measured in the follicular fluid by enzyme immunoassay. Injection of VEGF gene fragments increased the level of mRNA expression of VEGF 120 and 164 isoforms in the granulosa cells and VEGF protein contents in the follicular fluid. The number of preovulatory follicles and the capillary density in the theca interna increased significantly in the ovaries injected with VEGF gene fragments compared with those treated with eCG alone. The Flt-1, but not the Flk-1, mRNA expression show a tendency toward increasing in the thecal tissues of antral follicles in the ovaries injected with VEGF gene fragments. These results demonstrate that Flt-1 may be predominantly involved in the regulation of the capillary network in the theca interna during follicular development. Our data suggest that the regulation of perifollicular angiogenesis during follicular development is a very important factor in the development of ovulatory follicles. Our findings may offer an innovative technique for enhanced induction of follicular development in the ovary through gene and hormonal treatment, which may lead to prevention of infertility caused by ovarian dysfunction.

Although it is widely assumed that the cell type and genotype of the donor cell affect the efficiency of somatic cell cloning, little systematic analysis has been done to verify this assumption. The present study was undertaken to examine whether donor cell type, donor genotype, or a combination thereof increased the efficiency of mouse cloning. Initially we assessed the developmental ability of embryos that were cloned from cumulus or immature Sertoli cells with six different genotypes (i.e., 2 × 6 factorial). Significantly better cleavage rates were obtained with cumulus cells than with Sertoli cells (P < 0.005, two-way ANOVA), which probably was due to the superior cell-cycle synchrony of cumulus cells at G0/G1. After embryo transfer, there was a significant effect of cell type on the birth rate, with Sertoli cells giving the better result (P < 0.005). Furthermore, there was a significant interaction (P < 0.05) between the cell type and genotype, which indicates that cloning efficiency is determined by a combination of these two factors. The highest mean birth rate (10.8 ± 2.1%) was obtained with (B6 × 129)F1 Sertoli cells. In the second series of experiments, we examined whether the developmental ability of clones with the wild-type genotype (JF1) was improved when combined with the 129 genotype. Normal pups were cloned from cumulus and immature Sertoli cells of the (129 × JF1)F1 and (JF1 × 129)F1 genotypes, whereas no pups were born from cells with the (B6 × JF1)F1 genotype. The present study clearly demonstrates that the efficiency of somatic cell cloning, and in particular fetal survival after embryo transfer, may be improved significantly by choosing the appropriate combinations of cell type and genotype.

Avian perivitelline membrane, an investment homologous to the mammalian zona pellucida, is composed of at least two glycoproteins. Our previous studies demonstrated that one of its components, ZPC, which is synthesized in the ovarian granulosa cells, is secreted after carboxy-terminal proteolytic processing, and this event is a prerequisite event for ZPC secretion in quail. In the present study, we examined the role of the cytoplasmic tail, which is successfully removed after proteolytic processing, in membrane transport, proteolytic processing, and the secretion of quail ZPC. In pursuit of this, we produced a truncated ZPC mutant lacking the cytoplasmic tail located in its C-terminus and examined its expression in the mammalian cell line. Western blot analyses demonstrated that the cytoplasmic tail-deficient ZPC was neither secreted nor underwent proteolytic processing in the cells. Immunofluorescence analysis and the acquisition of resistance to endoglycosidase H digestion of the cytoplasmic tail-deficient ZPC demonstrated that the deletion of the cytoplasmic tail interferes with the intracellular trafficking of the protein from the endoplasmic reticulum to the Golgi apparatus. These results indicate that the cytoplasmic tail of quail ZPC might possess the determinant responsible for the efficient transport of the newly synthesized ZPC from the endoplasmic reticulum to the Golgi apparatus.

Progesterone (P) appears to stimulate sperm capacitation and/or induce the acrosome reaction (AR) in some species. In bovine, it is now well established that the BSP-A1/-A2 proteins (the major proteins of bovine seminal plasma) promote sperm capacitation. In this study, we investigated the effect of P on bovine sperm cholesterol efflux, capacitation, and the AR. Labeled bovine epididymal sperm were incubated (0–6 h) with different concentrations of P (0.01–10 μg/ml) in the presence or absence of BSP-A1/-A2 proteins (capacitating conditions). At different time intervals, aliquots of sperm were taken to determine the sperm cholesterol efflux, sperm capacitation (AR induced by lysophosphatidylcholine, lyso-PC), and sperm AR. The results show that the presence of P in the media did not affect the membrane cholesterol efflux potential of the BSP-A1/-A2 proteins. P alone did not stimulate the AR with or without lyso-PC unless the epididymal sperm were incubated in capacitating conditions (in the presence of BSP-A1/-A2). When washed ejaculated sperm were continuously incubated with P, the P did not stimulate AR. However, when ejaculated sperm were preincubated (6 h) with heparin (capacitation medium) and then incubated 15 min with P (2 μg/ml), the percentage of AR obtained was similar to that obtained with lyso-PC. The effect of P on sperm AR was concentration dependent with a maximum 2.2-fold increase at 2 μg/ml of P. These results demonstrate a potential role of P in bovine sperm AR but not in capacitation.

The present study investigated photorefractoriness in the prolactin (PRL) axis in hypothalamopituitary-disconnected (HPD) sheep exposed to prolonged long days. In experiment 1, HPD Soay rams transferred from short (8L:16D) to long (16L:8D) days for 48 wk to induce a cycle of activation, decline (photorefractoriness), and reactivation in PRL secretion were treated chronically with bromocriptine (dopamine-receptor agonist) or vehicle from the onset of photorefractoriness. Bromocriptine (0.01–0.04 mg kg⁻¹ day⁻¹; 12–24 wk of long days) blocked PRL release and caused a rebound response after the treatment, but it had no effect on the long-term PRL cycle (posttreatment PRL minimum, mean ± SEM, 35.3 ± 0.6 and 37.0 ± 0.4 wk for bromocriptine and control groups, respectively; not significant). In experiment 2, HPD rams were treated with sulpiride (dopamine-receptor antagonist) during photorefractoriness. Sulpiride (0.6 mg/kg twice daily; 22–30 wk of long days) induced a marginal increase in blood PRL concentrations, but again, it had no effect on the long-term PRL cycle (PRL minimum, 37.9 ± 0.4 and 37.6 ± 0.9 wk for sulpiride and control groups, respectively; not significant). The 24-h blood melatonin profile consistently reflected the long-day photoperiod throughout, and blood FSH concentrations were minimal, confirming the effectiveness of the HPD surgery. The results support the conclusion that photorefractoriness is regulated at the level of the pituitary gland independently of the PRL output signal.

The objective of this study was to examine the time during the postfertilization period that gene expression patterns in in vitro-cultured bovine embryos diverge from those of their in vivo-cultured counterparts. Presumptive bovine zygotes were produced by in vitro maturation and fertilization of immature oocytes collected from the ovaries of slaughtered animals. Approximately 20 h post insemination (hpi), zygotes were denuded and randomly divided into two groups for culture either in vitro, in synthetic oviduct fluid medium, or in vivo, in the ewe oviduct. Embryos were recovered from both systems at approximately 30 hpi (2-cell), 2 (4-cell), 3 (8-cell), 4 (16-cell), 5 (early morula), 6 (compact morula), or 7 (blastocyst) days post insemination. On recovery, they were examined for stage of development and snap frozen in liquid nitrogen for the analysis of transcript abundance using real-time polymerase chain reaction. The transcripts studied were glucose transporter 5, sarcosine oxidase, mitochondrial Mn-superoxide dismutase, connexin 43, interferon tau, insulin-like growth factor II, apoptosis regulator box-α and insulin-like growth factor-I receptor, most of which are known from our previous work to differ in terms of transcript abundance in blastocysts derived from culture in vitro or in vivo. The results demonstrate that the relative abundance of the transcripts studied varies throughout the preimplantation period and is strongly influenced by the culture environment. In addition, the data demonstrate that changes in transcript abundance in blastocyst stage embryos are in many cases a consequence of perturbed transcription earlier in development. Depending on the transcript, these differences may be evident by as little as 10 h of initiation of culture. Such information has implications not only for basic biology but also for human assisted reproduction in which there is a move toward culturing embryos to the blastocyst stage, necessitating prolonged culture in vitro under potentially deleterious conditions.

Calcitonin gene-related peptide (CGRP) and its related peptide, adrenomedullin (AM), are potent smooth muscle relaxants in a variety of tissues. The CGRP has been reported to play an important role in maintaining uterine relaxation during pregnancy. We have previously reported that CGRP-induced uterine relaxation was gestationally regulated. Calcitonin receptor-like receptor (CRLR), a seven-domain transmembrane protein functions as CGRP-A receptor, in association with receptor activity-modifying protein (RAMP) 1, a single-domain transmembrane protein, whereas CRLR and RAMP2 or RAMP3 constitute a receptor for AM. In the present investigation, we examined the mRNA expression of CRLR, RAMP1, RAMP2, and RAMP3 in rat uterus (n = 8) by reverse transcriptional analysis and polymerase chain reaction to assess the changes in the expression of CGRP-A- and AM-receptor components during pregnancy and labor and by steroid hormone treatments in adult ovariectomized rats. The changes in mRNA are expressed relative to the 18S mRNA in the uterus of rats at various stages: nonpregnant, pregnant on Day 18, spontaneous labor at term, Day 2 postpartum, and in pregnant rats on treatment with RU486. Ovariectomized rats treated for 3 days twice daily s.c. with estradiol-17β (2.5 μg/injection), progesterone (2 mg/injection), and the combination of estradiol-17β and progesterone (same doses as above) were also examined for the expression of various receptor components. Results showed that mRNA expression of the receptor components was significantly higher (P < 0.001 for CRLR, P < 0.01 for RAMP1, P < 0.05 for RAMP2, and P < 0.01 for RAMP3) in pregnant compared to nonpregnant rats. Except for RAMP3, expression of all the other three genes decreased significantly (P < 0.05) during labor. A progesterone antagonist, RU486 significantly decreased (P < 0.01 for CRLR, P < 0.05 for RAMP1, RAMP2, and RAMP3) all the receptor components during pregnancy. In adult ovariectomized rats, progesterone caused significant increases in CRLR (P < 0.001), RAMP1 (P < 0.05), and RAMP2 (P < 0.01). Levels of RAMP3 were unaffected by the progesterone treatment. Estradiol-17β treatment decreased all of the four receptor components significantly (P < 0.01 for CRLR, P < 0.05 for RAMP1, RAMP2, and RAMP3). Our results demonstrate that both CGRP and AM may play a role in uterine quiescence during pregnancy and that their receptor components are regulated by the steroid hormones.

Dendritic cells (DCs) in the pregnant human uterine mucosa have been poorly characterized, although they are likely to regulate immune responses to both placental trophoblast cells and uterine infections. In this study an HLA-DR , CD11c lin⁻ (CD3⁻, CD19⁻, CD56⁻, CD14⁻) population has been identified by three-color flow cytometry. The cell isolates were prepared either by collagenase digestion or mechanically from first-trimester decidual tissue. The decidual DCs comprised ∼1.7% of CD45 cells in the isolates and had the phenotype of immature myeloid DCs. No CD1a Langerhans cells or CD123 plasmacytoid DCs were detected. The decidual DCs were DC-SIGN⁻, DEC-205 , CD40 . Two subsets could be distinguished on the basis of relative expression of HLA-DR, which also differed in expression of DC-activation markers. The DCs were identified in situ by immunohistology by DEC-205 staining. Cells with dendritic processes were found scattered through both the decidua basalis (in which trophoblast cells are infiltrating) and the decidua parietalis. They were also visible in endothelial-lined spaces. This is the first study to identify and describe the phenotype and distribution of human decidual DCs.