BSP proteins and their homologs are a family of structurally related proteins characterized by the presence of tandem fibronectin type II domains. In the bovine species, BSP proteins were shown to be involved in sperm capacitation, a posttesticular maturation event necessary for sperm to acquire the ability to fertilize an oocyte. Recently, many new genes from this family have been discovered in numerous mammalian species. However, inconsistency in the nomenclature is creating much confusion. In light of the rapid growth of the BSP superfamily of proteins, we propose a new nomenclature in collaboration with the HUGO Gene Nomenclature Committee.

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)₂D), is a potent inducer of the antimicrobial protein cathelicidin, CAMP (LL37). In macrophages this response is dependent on intracrine synthesis of 1,25(OH)₂D from precursor 25-hydroxyvitamin D (25OHD), catalyzed by the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1). In view of the fact that trophoblastic cells also express abundant CYP27B1, we postulated a similar intracrine pathway for induction of CAMP in the placenta. Analysis of placenta explants, primary cultures of human trophoblast, and the 3A trophoblastic cell line treated with 1,25(OH)₂D (1–100 nM) revealed dose-dependent induction of CAMP similar to that observed with primary cultures of human macrophages. Also consistent with macrophages, induction of trophoblastic CAMP was enhanced via intracrine conversion of 25OHD to 1,25(OH)₂D. However, in contrast to macrophages, induction of CAMP by vitamin D in trophoblasts was not enhanced by costimulation with Toll-like receptor ligands, such as lipopolysaccharide. Despite this, exposure to vitamin D metabolites significantly enhanced antibacterial responses in trophoblastic cells: 3A cells infected with Escherichia coli showed decreased numbers of bacterial colony-forming units compared with vehicle-treated controls when treated with 25OHD (49.6% ± 10.9%) or 1,25(OH)₂D (45.4% ± 9.2%), both P < 0.001. Treatment with 25OHD (1–100 nM) or 1,25(OH)₂D (0.1–10 nM) also protected 3A cells against cell death following infection with E. coli (13.6%–26.9% and 22.3%–40.2% protection, respectively). These observations indicate that 1,25(OH)₂D can function as an intracrine regulator of CAMP in trophoblasts, and may thus provide a novel mechanism for activation of innate immune responses in the placenta.

Two mouse models of pelvic organ prolapse have been generated recently, both of which have null mutations in genes involved in elastic fiber synthesis and assembly (fibulin 5 and lysyl oxidase-like 1). Interestingly, although these mice exhibit elastinopathies early in life, pelvic organ prolapse does not develop until later in life. In this investigation we developed and validated a tool to quantify the severity of pelvic organ prolapse in mice, and we used this tool prospectively to study the role of fibulin 5, aging, and vaginal proteases in the development of pelvic organ prolapse. The results indicate that >90% of Fbln5^−/− mice develop prolapse by 6 mo of age, even in the absence of vaginal delivery, and that increased vaginal protease activity precedes the development of prolapse.

Leptin plays a role in both energy homeostasis and reproduction, and it is required in early pregnancy. It stimulates metalloproteinase activity in cultured human trophoblasts and invasiveness of cultured mouse trophoblasts. Our goal has been to examine mechanisms that underpin the ability of leptin to promote trophoblast invasiveness in primary cultures of mouse trophoblasts. Leptin stimulated the phosphorylation of MEK (MAP2K1) but not signal transducer and activator of transcription 3 (STAT3) in the cultures, increased the concentration of the suppressor of cytokine signaling 3 (SOCS3) protein, and upregulated metalloproteinase activity. Microarray analysis revealed that leptin stimulated select genes with roles in cell motility, including Stmn, a gene linked to invasiveness in other cell types. There was also an increase in activity of several genes associated with MAPK and RhoGTPase signaling. In addition, leptin muted expression of genes correlated with terminal differentiation of trophoblast giant cells, including ones associated with the TGFbeta signaling pathway and endoreduplication of DNA, and upregulated selected prolactin-related family members. Feulgen staining of leptin-treated cells revealed a loss of cells with low ploidy. The data suggest that leptin accelerates disappearance of non-giant cells while inhibiting terminal differentiation of committed giant cells, possibly by maintaining cells in an intermediate stage of differentiation.

Female mice treated neonatally with the phytoestrogen genistein (50 mg/kg/day) have multioocyte follicles, lack regular estrous cyclicity, and are infertile even after superovulation. To determine the cause of their infertility, we examined oocyte developmental competence and timing of embryo loss. Eggs obtained by superovulation of genistein-treated or control females were equally capable of being fertilized in vitro and cultured to the blastocyst stage. However, if eggs were fertilized in vivo, retrieved at the pronucleus stage, and cultured, there was a significant reduction in the percentage of embryos from genistein-treated females reaching the blastocyst stage. When these blastocysts were transferred to pseudopregnant recipients, the number of live pups produced was similar to that in controls. Preimplantation embryo development in vivo was examined by flushing embryos from the oviduct and/or uterus. Similar numbers of one-cell and two-cell embryos were obtained from genistein-treated and control females. However, significantly fewer embryos (<50%) were obtained from genistein-treated females on postcoital Days 3 and 4. To determine if neonatal genistein treatment altered the ability of the uterus to support implantation, blastocysts from control donors were transferred to control and genistein-treated pseudopregnant recipients. These experiments demonstrated that genistein-treated females are not capable of supporting normal implantation of control embryos. Taken together, these results suggest that oocytes from mice treated neonatally with genistein are developmentally competent; however, the oviductal environment and the uterus have abnormalities that contribute to the observed reproductive failure.

Folliculogenesis is a coordinated process, and the genes that regulate development are difficult to investigate in vivo. In vitro culture systems permit the assessment of individual follicles during development, thereby enabling gene expression patterns to be monitored during follicle development. Mouse multilayered secondary follicles (150–180 μm in diameter) were cultured in three-dimensional matrices of varying physical properties for up to 8 days. During this period of follicle growth in vitro, antrum formation and steroid production were monitored, and mRNA was isolated. The expression levels of genes (Star, Cyp11a1, Cyp17a1, Hsd3b1, Cyp19a1, Fshr, Lhcgr, Aqp7, Aqp8, Aqp9, and Hif1a) were measured and correlated to follicle developmental status. Follicles that developed an antrum and produced appropriate levels of estrogen and progesterone had unchanging expression of Star, Aqp7, Aqp8, and Hif1a and a 34-fold increase in Cyp19a1 expression at Day 8 of culture and had elevated Lhcgr at Days 6 and 8 of culture. Follicles that were healthy but did not form an antrum or produce appropriate levels of steroids, however, demonstrated increasing levels of Star, Aqp7, Aqp8, and Hif1a and a 15-fold increase in Cyp19a1 at Day 8 of culture, and Lhcgr levels were not elevated until Day 8 of culture. To our knowledge, this study provides the first temporal analysis of gene expression using individual culture in alginate hydrogels that correlates growth and steroidogenesis during follicle development and identifies expression patterns in healthy follicles and in developmentally disadvantaged follicles.

Previous studies demonstrated that maternal cocaine administration caused a significant decrease in protein kinase C epsilon (PRKCE) abundance in the left ventricle and an increase in susceptibility of the heart to ischemic injury in adult male offspring. The present study tested the hypothesis that epigenetic modification has a key role in cocaine-mediated programming of cardiac Prkce gene repression. Pregnant Sprague-Dawley rats were administered saline or cocaine (30 mg/kg/day i.p.) from Days 15 to 21 of gestational age, and hearts of 3-mo-old adult offspring were studied. Cocaine exposure significantly decreased Prkce mRNA levels in the left ventricle of male but not female offspring. CpG dinucleotides identified in Bhlhb2, Pparg, E2f, and Egr1 binding sites at the Prkce gene promoter were densely methylated in males and females and were unaffected by cocaine exposure. In contrast, methylation of CpGs in the two Sp1 binding sites (−346 and −268) was low and was significantly increased by cocaine exposure in male offspring. In females, methylation of the Sp1 binding site at −268 but not −346 was increased. Reporter gene assays showed that both Sp1 binding sites had a strong stimulatory role in Prkce gene activity. Methylation of the Sp1 binding sites significantly decreased SP1 binding to the Prkce promoter. Cocaine exposure did not affect nuclear SP1 protein levels but decreased the SP1 binding affinity to its binding site at −268. The results demonstrate an epigenetic mechanism of DNA methylation in programming of cardiac Prkce gene repression, linking fetal cocaine exposure and pathophysiological consequences in the heart of adult male offspring in a gender-dependent manner.

A conserved feature of germ cell cytokinesis is the formation of stable intercellular bridges between daughter cells. These intercellular bridges are seen in diverse species from Drosophila melanogaster to Homo sapiens and have been shown to have roles in communication of large numbers of germ cells. In testis expressed gene 14 (Tex14) knockout mice, intercellular bridges do not form during spermatogenesis, and male mice are sterile, demonstrating an essential role for intercellular bridges in postnatal spermatogenesis in mammals. Intercellular bridges also form between dividing germ cells in both male and female embryos. However, little is known about the formation or role of the embryonic intercellular bridges in mammals. In females, embryonic intercellular bridges have been proposed to have a role in development of the presumptive oocyte. Herein, we show that TEX14 is an essential component of male and female embryonic intercellular bridges. In addition, we demonstrate that mitotic kinesin-like protein 1 (MKLP1, official symbol KIF23), which we have discovered is a component of intercellular bridges during spermatogenesis, is also a component of male and female embryonic intercellular bridges. Germ cell intercellular bridges are readily identified by KIF23 immunofluorescence between the gonocytes and oogonia of control mice but are absent between germ cells of Tex14-null mice. Furthermore, by electron microscopy, intercellular bridges are present in all control newborn ovaries but are absent in the Tex14 knockout ovaries. Despite the absence of embryonic intercellular bridges in the Tex14-null mice, male mice initiate spermatogenesis, and female mice are fertile. Although fewer oocytes were present in Tex14-null neonatal ovaries, folliculogenesis was still active at 1 yr of age. Thus, while TEX14 and intercellular bridges have an essential role in postnatal spermatogenesis, they are not required in the embryo.

Two experiments were conducted to test the hypothesis that cortisol interferes with the positive feedback action of estradiol that induces the luteinizing hormone (LH) surge. Ovariectomized sheep were treated sequentially with progesterone and estradiol to create artificial estrous cycles. Cortisol or vehicle (saline) was infused from 2 h before the estradiol stimulus through the time of the anticipated LH surge in the artificial follicular phase of two successive cycles. The plasma cortisol increment produced by infusion was ∼1.5 times greater than maximal concentrations seen during infusion of endotoxin, which is a model of immune/inflammatory stress. In experiment 1, half of the ewes received vehicle in the first cycle and cortisol in the second; the others were treated in reverse order. All ewes responded with an LH surge. Cortisol delayed the LH surge and reduced its amplitude, but both effects were observed only in the second cycle. Experiment 2 was modified to provide better control for a cycle effect. Four treatment sequences were tested (cycle 1-cycle 2): vehicle-vehicle, cortisol-cortisol, vehicle-cortisol, cortisol-vehicle. Again, cortisol delayed but did not block the LH surge, and this delay occurred in both cycles. Thus, an elevation in plasma cortisol can interfere with the positive feedback action of estradiol by delaying and attenuating the LH surge.

New techniques to boost male and female fertility are being pioneered at a rapid pace in fertility clinics to increase the efficiency of assisted reproduction methods in couples in which natural conception has not been achieved. This study investigates the possible epigenetic effects of ooplasm manipulation methods on postnatal growth and development using a mouse genetic model, with particular emphasis on the possible effects of intergenotype manipulations. We performed interstrain and control intrastrain maternal pronuclear transfers, metaphase-II spindle transfers, and ooplasm transfer between C57BL/6 and DBA/2 mice, and found no major, long-term growth defects or epigenetic abnormalities, in either males or females, associated with intergenotype transfers. Ooplasm transfer itself was associated with reduced viability, and additional subtle effects of ooplasm strain of origin were observed. Both inter- and intrastrain ooplasm transfer were associated with subtle, transient effects on growth early in life. We also performed inter- and intrastrain germinal vesicle transfers (GVTs). Interstrain GVT females, but not males, had significantly lower body weights at birth and thereafter compared with the intrastrain GVT and non-GVT controls. No GVT-associated changes were observed in DNA methylation of the Mup1, Rasgrf1, H19, Snrpn, or Peg3 genes, nor any difference in expression of the imprinted Rasgrf1, Igf2r, or Mest genes. These results indicate that some ooplasm manipulation procedures may exert subtle effects on growth early in life, while intergenotype GVT can result in significant growth deficiencies after birth.

In mammals, circadian genes, Clock, Arntl (also known as Bmal1), Cry1, Cry2, Per1, Per2, and Per3, are rhythmically transcribed every 24 h in almost all organs and tissues to tick the circadian clock. However, their expression and function in oocytes and preimplantation embryos have not been investigated. In this study we found that the circadian clock may stop in mouse oocytes and preimplantation embryos. Real-time PCR analysis revealed the presence of transcripts of these genes in both oocytes and preimplantation embryos; however, their amounts did not oscillate every 24 h in one- to four-cell and blastocyst-stage embryos. Moreover, immunofluorescence analyses revealed that CLOCK, ARNTL, and CRY1 were localized similarly in the nuclei of germinal vesicle (GV) oocytes and one-cell- to four-cell-stage embryos. Because CRY1 is known to interact with the CLOCK-ARNTL complex to suppress transcription-promoting activity of the complex for genes such as Wee1, Cry2, Per1, Per2, and Per3 in cells having the ticking circadian clock, we hypothesized that if the circadian clock functions in GV oocytes and one-cell- to four-cell-stage embryos, CLOCK, ARNTL, and CRY1 might suppress the transcription of these genes in GV oocytes and one-cell- to 4-cell-stage embryos as well. As a result, knockdown of CRY1 in GV oocytes by RNA interference did not affect the transcription levels of Wee1, Cry2, Per1, Per2, and Per3, but it reduced maturation ability. Thus, it seems that circadian genes are not involved in circadian clock regulation in mouse oocytes and preimplantation embryos but are involved in physiologies, such as meiosis.

Prior studies have demonstrated that combined treatment of testosterone with a progestin induces a more rapid and greater suppression of spermatogenesis than testosterone treatment alone. We hypothesized that the suppressive effects of the combination of testosterone undecanoate (TU) injections plus oral levonorgestrel (LNG) on spermatogenesis may be mediated through a greater perturbation of testicular gene expression than TU alone. To test this hypothesis, we performed open testicular biopsy on 12 different adult healthy subjects: 1) four healthy men as controls; 2) four men 2 wk after TU treatment; and 3) four men 2 wk after TU LNG administration. RNA isolated from biopsies was used for DNA microarray using the Affymetrix Human Genome U133 Plus 2.0 oligonucleotide microarrays. Gene expression with ≥2-fold changes (P < 0.05) compared with control was analyzed using the National Institutes of Health Database for Annotation, Visualization, and Integrated Discovery 2008 resource. The TU treatment altered the gene expression in 109 transcripts, whereas TU LNG altered the gene expression in 207 transcripts compared with control. Both TU and TU LNG administration suppressed gene expression of insulin-like 3; cytochrome P450, family 17, subfamily A1 in Leydig cells; and inhibin alpha in Sertoli cells; they increased proapoptotic transcripts BCL2-like 14, insulin-like growth factor-binding protein 3; and they decreased X-linked inhibitor of apoptosis protein. In comparison with TU treatment alone, TU LNG treatment upregulated insulin-like 6 and relaxin 1, and downregulated RNA-binding protein transcripts. We conclude that TU LNG administration induces more changes in testicular gene expression than TU alone. This exploratory study provided a novel and valuable database to study the mechanisms of action of hormonal regulation of spermatogenesis in men and identified testicular-specific molecules that may serve as potential targets for male contraceptive development.

We examined whether impairment of intracellular Ca² homeostasis is related to poor embryo development in in vitro-aged oocytes. We found that in vitro aging of mouse oocytes affected the patterns of Ca² oscillations at fertilization: these Ca² oscillations were lower in amplitude and higher in frequency compared with oocytes without in vitro aging. We also observed that the intracellular Ca² store was decreased in in vitro-aged oocytes. A decrease in the Ca² store induced by thapsigargin, a specific endoplasmic reticulum (ER) membrane Ca² -ATPase inhibitor, resulted in a lower fertilization rate and in poorer embryo development. The frequency of Ca² oscillations was significantly increased at fertilization, whereas their amplitude was decreased in thapsigargin-treated oocytes. These results suggest that impairment of intracellular Ca² homeostasis (such as a decrease in the ER Ca² store) caused an alteration in Ca² oscillations and the poor embryo development in in vitro-aged oocytes. Because embryo fragmentation is closely related to apoptosis, we examined expression of BAX (a proapototic protein) and BCL2 (an antiapoptotic protein) in in vitro-aged oocytes. Although BCL2 was strongly expressed in oocytes without in vitro aging, expression of BCL2 was significantly reduced in oocytes of other culture conditions and treatments such as those in in vitro aging and those that were pretreated with H₂O₂ or thapsigargin. Acting together, alteration in Ca² oscillations and decrease in BCL2 expression in in vitro-aged oocytes may lead to poor embryo development.

In rats, the success of in vitro fertilization (IVF) was reported 40 years ago. Although it has been demonstrated in papers that these IVF oocytes using sperm freshly collected from cauda epididymides can be developed to term via embryo transfer, successful IVF with cryopreserved rat sperm has never been reported to date. Here, we report establishment of a successful IVF system using frozen/thawed rat spermatozoa. Our data showed that intracellular cAMP and free cholesterol levels in frozen/thawed rat sperm were maintained low, suppressing capacitation-associated tyrosine phosphorylation. The treatment of methyl-beta-cyclodextrin improved removal of free cholesterol from the membrane in frozen/thawed sperm but not induction of capacitation-associated tyrosine phosphorylation in the sperm. Treatment with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX), dramatically increased cAMP and tyrosine phosphorylation levels in frozen/thawed rat sperm. When the IBMX-treated frozen/thawed sperm were used for IVF, the proportions of pronuclear formation and blastocyst formation were significantly higher than those of frozen/thawed sperm treated without IBMX (P < 0.05). The embryos were developed to term at a high success rate equivalent to the rate obtained with IVF using fresh sperm. Thus, we established for the first time a successful IVF system in rats using cryopreserved spermatozoa.

Spermiogenesis is a complex process consisting of three main phases: the round, elongating, and elongated spermatid phases. Although the germ cells acquire a haploid set of paternal chromosomes after meiosis, how functional these male haploid germ cells are as male gametes at various differentiation stages has remained unclear. We selectively injected specific steps of haploid male germ cells into oocytes and assessed the function of the zygotes. Applying the transillumination technique using acrosin-green fluorescent protein transgenic mice, we succeeded in selecting four types of haploid male germ cells for microinsemination: early round spermatids (steps 2–3), late round spermatids (steps 7–8), elongating spermatids (steps 9–10), and elongated spermatids (step 16). The microinsemination technique revealed that the early and late round spermatids had similar developmental abilities in producing progeny, indicating that the nuclear status of newly generated round spermatids was similar to that of late round spermatids. An increased birthrate of progeny was first observed in steps 9–10 of elongating spermatids, but the frequency was slightly lower than that of the elongated spermatids. These results indicated that the transition from steps 7–8 of round spermatids to steps 9–10 of elongating spermatids is a key step in changing the nuclear status of male gametes in producing progeny.

Male germ cells undergo dynamic epigenetic reprogramming during fetal development, eventually establishing spermatogonial stem cells (SSCs) that can convert into pluripotent stem cells. However, little is known about the developmental potential of fetal germ cells and how they mature into SSCs. We developed a culture system for fetal germ cells that proliferate for long periods of time. Male germ cells from embryos 12.5–18.5 days postcoitum could expand by glial cell line-derived neurotrophic factor, a self-renewal factor for SSCs. These cells did not form teratomas, but repopulated seminiferous tubules and produced spermatogenesis, exhibiting spermatogonia potential. However, the offspring from cultured cells showed growth abnormalities and were defective in genomic imprinting. The imprinting defect persisted in both the male and female germlines for at least four generations. Moreover, germ cells in the offspring showed abnormal histone modifications and DNA methylation patterns. These results indicate that fetal germ cells have a limited ability to become pluripotent cells and lose the ability to undergo epigenetic reprogramming by in vitro culture.

Epidermal growth factor (EGF) has been shown to stimulate survival in diverse cells in vitro. In the present study, the effects of EGF and the EGF-related signaling pathway on proliferation of chicken primordial germ cells (PGCs) were investigated. Results showed that EGF (10–100 ng/ml) increased the number and area of PGC colonies in a time- and dose-dependent manner. EGF also activated PKC, a process that was inhibited by AG1478 (an EGFR tyrosine kinase inhibitor) and ethyleneglycol-bis-(beta-aminoethyl ether)-N,N′-tetraacetic acid (EGTA; an intracellular Ca² chelator). In addition, the degradation of NFKBIA and NFKB1 (p65) translocation was observed after EGF treatment, which was significantly blocked by pretreatment with AG1478, EGTA, H₇, or SN50 (NFKB1-specific inhibitor). Furthermore, we found that EGF-induced cell proliferation was significantly attenuated by AG1478, EGTA, H₇, and SN50, respectively. On the other hand, inhibition of EGFR, Ca² /PKC, or NFKB1 abolished the EGF-stimulated increase in the expression of cyclins CCND1 and CCNE1, cyclin-dependent kinase 6 (CDK6), CDK2, and BCL2, and restored the EGF-induced inhibition of BAX expression and caspase 3/9 activity, indicating that EGFR, PKC, and NFKB1 signaling cascades were involved in EGF-stimulated DNA synthesis and antiapoptosis action. In conclusion, EGF stimulated proliferation of chicken PGCs via activation of Ca² /PKC involving NFKB1 signaling pathway. These observations suggest that EGF signaling is important in regulating germ cell proliferation in the chicken embryonic gonad.

The Nile tilapia (Oreochromis niloticus) is economically one of the most important freshwater fish and is an excellent model for studies under laboratory conditions. Temperature is considered a very important modulator of reproductive activity in fish, although few studies have specifically addressed the effects of this key factor on morphological and functional aspects of teleost testes. Therefore, our main objectives in the present study were to analyze the effects of different temperatures (20, 25, 30, and 35°C) on testicular somatic and germ cells in sexually mature Nile tilapias. Compared with fish kept at other temperatures, tilapias maintained at 20°C demonstrated increased (P < 0.05) Sertoli cell and Leydig cell proliferation, volume density and frequency of most type B spermatogonia, and germ cell apoptosis. Conversely, tubular fluid secretion was decreased (P < 0.05) in the same animals. Although not significant, type A spermatogonia proliferation followed the pattern established for Sertoli cell and Leydig cell mitotic activity, suggesting that they preferentially would proliferate at lower temperatures. Based on most results found in our study and considering that tilapias are nonseasonal breeders, we suggest a model for temperature action on tilapia testes in which lower temperature (20°C) would favor type A spermatogonial renewal, Sertoli cell and Leydig cell proliferation, and germ cell apoptosis, whereas higher temperatures (30–35°C) would trigger rapid germ cell differentiation. Thus, tilapias could potentially be utilized in studies involving hormones and factors related to Sertoli cell and Leydig cell proliferation and spermatogonial self-renewal or differentiation.

Mouse spermatogenic cells are known to contain at least two isoforms of cytoplasmic poly(A)-binding proteins, PABPC1 and PABPC2 (previously known as PABPT). In this study, we have characterized PABPC1 and PABPC2. PABPC2 was present in pachytene spermatocytes and round spermatids, whereas elongating spermatids still included PABPC1. These two proteins are capable of binding mRNA poly(A) tails nonspecifically and of directly associating with each other and with several translational regulators, including EIF4G1, PAIP1, PAIP2, and PIWIL1 (previously known as MIWI). Moreover, both PABPC1 and PABPC2 exhibited the ability to enhance translation of a reporter mRNA in vitro. Despite these similarities, PABPC2 was distinguished from PABPC1 by the absence of PABPC2 in actively translating polyribosomes of testicular cells. PABPC1 was distributed in polyribosomes and in translationally inactive messenger ribonucleoprotein particles. Most importantly, PABPC2 and PIWIL1 were noticeably enriched in the chromatoid body of round spermatids. These results suggest that PABPC2 may function in translational repression during spermatogenesis.

Mouse oocytes undergo two successive meiotic divisions to generate one large egg with two small polar bodies. The divisions are essential for preserving the maternal resources to support embryonic development. Although previous studies have shown that some small guanosine triphosphatases, such as RAC, RAN, and CDC42, play important roles in cortical polarization and spindle pole anchoring, no oocytes undergo cytokinesis when the mutant forms of these genes are expressed in mouse oocytes. Here, we show that the ADP-ribosylation factor 1 (ARF1) plays an important role in regulating asymmetric cell division in mouse oocyte meiosis. Microinjection of mRNA of a dominant negative mutant form of Arf1 (Arf1^T31N) into fully grown germinal vesicle oocytes led to symmetric cell division in meiosis I, generating two metaphase II (MII) oocytes of equal size. Subsequently, the two MII oocytes of equal size underwent the second round of symmetric cell division to generate a four-cell embryo (zygote) when activated parthenogenetically or via sperm injection. Furthermore, inactivation of mitogen-activated protein kinase (MAPK) but not MDK (also known as MEK) has been discovered in the ARF1 mutant oocytes, and this further demonstrated that ARF1, MAPK pathway plays an important role in regulating asymmetric cell division in meiosis I. Similarly, ARF1^T31N-expressing, superovulated MII oocytes underwent symmetric cell division in meiosis II when activation was performed. Rotation of the MII spindle for 90 degrees was prohibited in ARF1^T31N-expressing MII oocytes. Taken together, our results suggest that ARF1 plays an essential role in regulating asymmetric cell division in female meiosis.

It is still difficult to successfully cryopreserve in vitro-produced (IVP) swine embryos, as they are sensitive to chilling due to the abundance of intracellular lipids. Mechanical delipation through micromanipulation is successful, but this method increases the potential of pathogen transmission because of the damage inflicted upon the zona pellucida during micromanipulation, and it is labor intensive. Reported here is a method to remove the lipid of IVP porcine embryos, without significantly compromising the zona pellucida, by trypsin treating the embryos or exposing the embryo to a high-osmolality solution to enlarge the perivitelline space so that the lipid could be polarized and separated completely after subsequent centrifugation without micromanipulation. The procedures work both for nuclear transfer-derived embryos and in vitro-fertilized embryos. Both methods provide a high-throughput process that leaves the zona pellucida intact (or relatively intact for the trypsin treatment) to aid in preventing disease transmission. It is also demonstrated that this procedure results in viable piglets, a claim that could not be made in many previous reports. Although the efficiencies of cryopreservation have not been dramatically improved, these procedures allow a single person to process very large numbers of embryos without the necessity of manipulating each individual embryo on a micromanipulator. Such high-throughput processing overcomes the lack of high efficiency (i.e., the system can be overloaded with embryos for transfer to surrogates).

During spermiogenesis of an alga Chara vulgaris, which in many aspects resembles that of animals, histones are replaced by protamine-type proteins. Our earlier immunocytochemical studies showed that this replacement started during the short stage V of spermiogenesis, when electronograms revealed an extensive system of cisternae and vesicles of endoplasmic reticulum (ER). The present studies revealed at stage V intensive incorporation of labeled ³H-arginine and ³H-lysine quickly translocating into a nucleus visualized with pulse-chase autoradiography of semithin sections. The immunogold technique with the use of the antibodies to protamine-type proteins isolated from Chara tomentosa show that both ER cisternae and vesicles are labeled with gold grains, which are absent from the spermatids not treated with the antibodies; thus, the ER is probably the site of the protamine-type protein synthesis. These proteins then are translocated to a nucleus through ER channels connected with the nuclear envelope, as suggested by gold labeling of an inner membrane of the nuclear envelope adjacent to condensed chromatin. The above results correspond with those of other authors showing that in animals, protamines bind with lamin B receptors localized in the inner membrane of the nuclear envelope. A hypothesis has been put forward that during Chara spermiogenesis the inner membrane of the nuclear envelope invaginates into a nucleus together with protamine-type proteins, which become separated from the membrane and penetrate into chromatin.

Mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury in peripubertal rodents results in the stimulation of germ cell apoptosis through an interaction of FAS/FASL between these two cell types. During this peripubertal period, an early spike in the incidence of germ cell apoptosis occurs during the first wave of spermatogenesis and is essential for the development of functional spermatogenesis in adults. Our previous observations revealed that soluble tumor necrosis factor alpha (sTNFA) released by germ cells after MEHP exposure consequently resulted in a robust induction of FASL by Sertoli cells. Metalloproteinases (MPs) are essential for processing the TNFA precursor to its soluble form and its ability to bind to TNFRSF1A. The activity of MPs is regulated by the tissue inhibitors of MPs (TIMPs) family. Herein we report that TIMP2 is predominately expressed in Sertoli cells and that protein levels decrease in a time-dependent manner after MEHP exposure. The secretion of matrix MP 2 (MMP2) in primary rat Sertoli cell-germ cell cocultures is induced after MEHP exposure, and its activity increases in a time-dependent manner. The addition of SB-3CT, a specific gelatinase inhibitor, decreases the activity of MMP2 and significantly reduces MEHP-enhanced sTNFA production in primary cocultures. In vivo challenges with SB-3CT decrease sTNFA and reduce MEHP-induced testicular germ cell apoptosis. In primary cocultures, MEHP exposure causes a 9.46-fold increase in sTNFA, while the addition of recombinant MMP2 protein results in a 5.4-fold increase in sTNFA, suggesting that MEHP-induced MMP2 is in part responsible for the activation of TNFA in the testis. Taken together, these observations indicate the distinct role of specific MPs in response to toxicant-induced Sertoli cell injury, providing further insights into the mechanism by which Sertoli cells control the sensitivity of germ cells to undergo apoptosis.

During spermatogenesis, leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) to gain entry into the adluminal compartment for further development. At the same time, these as well as other germ cell types in the epithelium must retain their close association with Sertoli cells via specialized cell junctions. In this study, we demonstrate that RAB13—a guanosine triphosphatase (GTPase) known to participate in tight junction function in other epithelia—also participates in the dynamics of the ectoplasmic specialization, a testis-specific type of anchoring junction. By immunohistochemistry microscopy, RAB13 localized to the ectoplasmic specialization. Moreover, RAB13 was found to associate with vinculin (VCL) and espin (ESPN), two putative ectoplasmic specialization actin (ACT)-binding proteins, by coimmunoprecipitation and immunofluorescence microscopy experiments. To address the role of RAB13 in ectoplasmic specialization dynamics, an in vivo model was used in which administration of Adjudin induced the disassembly of Sertoli-germ cell anchoring junctions. Following administration of this drug, the RAB13 level decreased steadily when the loss in testicular weight was taken into account. Similarly, the association of RAB13 with VCL decreased but was not completely lost during Adjudin-mediated ectoplasmic specialization restructuring. Taken collectively, these results suggest that RAB13 functions in ectoplasmic specialization dynamics in the testis.

Arginine is an essential amino acid for conceptus (embryo/fetus and trophoblast/placenta) growth and development; however, the mechanisms for arginine transport into the uterine lumen and uptake by conceptuses are largely unknown. In this study, expression of System y (SLC7A1, SLC7A2, and SLC7A3) cationic amino acid transporters in uteri of cyclic and pregnant ewes and conceptuses was studied, and effects of pregnancy, progesterone (P4), and interferon tau (IFNT) on their expression were investigated. SLC7A1 mRNA was most abundant in endometrial luminal (LE) and superficial glandular (sGE) epithelia on Day 16 of the estrous cycle and on Days 16–20 of pregnancy, whereas SLC7A2 mRNA was most abundant in LE and mid to deep glandular (GE) epithelia on Days 14–20 of gestation. Expression of SLC7A1 and SLC7A2 was enhanced in pregnant ewes in a cell-specific manner, but abundance of SLC7A3 was not affected by day of the estrous cycle or by pregnancy status. SLC7A1, SLC7A2, and SLC7A3 mRNAs were expressed in trophectoderm and endoderm of conceptuses. In ovariectomized ewes, short-term treatment of ewes with P4 and IFNT did not affect endometrial SLC7A1 mRNA, while long-term treatment with P4 stimulated SLC7A1 in LE and GE, and IFNT tended to increase SLC7A1 abundance in LE. SLC7A2 mRNA abundance increased 4.1-fold in response to short-term P4 treatment and an additional 1.7-fold by IFNT primarily in endometrial LE/sGE, and these effects were ablated by a P4 receptor antagonist. These results indicate that coordinate changes in SLC7A1, SLC7A2, and SLC7A3 expression in uterine endometria and conceptuses are likely important in transport of arginine that is critical to conceptus growth, development, and survival.