My interest in germ cells began when I first witnessed sea urchin fertilization and embryo development during a laboratory class at Hokkaido University, Sapporo, Japan, almost 60 yr ago. Weismann's concept of germ cells that I learned during my undergraduate years became the driving force of my entire research career. During the early years, my associates and I used mainly the golden hamster and the guinea pig as model animals because their spermatozoa had large acrosomes and we could readily follow changes in the acrosomes without killing or staining spermatozoa. We later used the mouse as our model organism because we wanted to produce live offspring with known genetic backgrounds. A summary of the findings we made during those years includes the following: (1) first in vitro sperm capacitation; (2) discovery of sperm hyperactivation; (3) demonstration of the importance of Ca² in sperm acrosome reaction, hyperactivation, sperm-egg fusion, and egg activation; (4) development of the sperm's fusion competence during the acrosome reaction; (5) characterization of sperm-oviduct relationships before and during fertilization; (6) use of zona-free hamster eggs to examine fertilizing ability and chromosomes of human spermatozoa; (7) development and use of intracytoplasmic sperm injection; (8) use of prespermatozoal cells for the production of offspring; (9) sperm preservation by freeze-drying and freezing whole-animal bodies; and (10) mouse cloning by somatic cell nuclear transfer. My current interests and my visions for the future include the following: (1) mass production of mature eggs and spermatozoa in vitro, (2) permanent sperm preservation at ambient temperature, (3) development of safer and more efficient assisted fertilization (reproduction) technologies, (4) development of safe and efficient methods of cloning, (5) production of artificial organs such as an artificial uterus, (6) development of safe and effective male contraceptives, and (7) prevention of cancer through germ cell research.

This study investigated the nucleotide sequences and tissue expression levels of genes relating to the ovulation rate in Yunling black goats, a famous Yunnan province, China, local breed with low fecundity. Five genes, FSHB, FSHR, BMP15, BMPR1B, and ESR2, were investigated; the complete cDNA sequences of these genes were 390, 2088, 1185, 1509, and 1585 bp, respectively, and compared with Boer goats (a more fecund breed), the sequence identities were 99%, 99%, 99%, 100%, and 99%, respectively. There were two base differences in FSHB and BMP15, four in FSHR, and three in ESR2. There were fewer follicles and oocytes in Yunling black goats than in Boer goats. Expression levels of FSHB, FSHR, and BMP15 genes in Yunling black goats were lower, and expression levels of BMPR1B and ESR2 genes were higher. Serum FSH content was lower in Yunling black goats, but serum estrogen content was higher. Protein expression levels of FSHR, BMP15, BMPR1B, and ESR2 in ovary and pituitary correlated positively with gene mRNA expression levels. In Yunling black goats, the mRNA expression levels of FSHB, FSHR, and BMP15 positively correlated with litter size, but those of BMPR1B and ESR2 correlated negatively. Together, base changes and variations in mRNA and protein expression levels of genes relating to the ovulation rate result in low fecundity in the Yunling black goat. Reduced BMP15 and FSHR levels may be related to the observed fewer oocytes and, consequently, fewer follicles.

The objective of this study was to identify transcription factors associated with differentiation of the chorionic girdle, the invasive form of equine trophoblast. The expression patterns of five transcription factors were determined on a panel of conceptus tissues from early horse pregnancy. Tissues from Days 15 through 46 were tested. Eomesodermin (EOMES), glial cells missing homologue 1 (GCM1), heart and neural crest derivatives expressed transcript 1 (HAND1), caudal type homeobox 2 (CDX2), and distal-less homeobox 3 (DLX3) were detected in horse trophoblast, but the expression patterns for these genes varied. EOMES had the most restricted distribution, while DLX3 CDX2, and HAND1 were widely expressed. GCM1 seemed to increase in the developing chorionic girdle, and this was confirmed by quantitative RT-PCR assays. GCM1 expression preceded a striking increase in expression of equine chorionic gonadotropin beta (CGB) in the chorionic girdle, and binding sites for GCM1 were discovered in the promoter region of the CGB gene. GCM1, CGB, and CGA mRNA were expressed preferentially in binucleate cells as opposed to uninucleate cells of the chorionic girdle. Based on these findings, it is likely that GCM1 has a role in differentiation and function of the invasive trophoblast of the equine chorionic girdle and endometrial cups. The equine binucleate chorionic girdle (CG) secreting trophoblast shares molecular, morphological, and functional characteristics with human syncytiotrophoblast and represents a model for studies of human placental function.

Marijuana is the most commonly used illicit drug. Although there is some indication that reproductive functions in males are impaired in chronic marijuana users, the genetic evidence and underlying causes remain largely unknown. Herein we show that genetic loss of Faah, which encodes fatty acid amide hydrolase (FAAH), results in elevated levels of anandamide, an endocannabinoid, in the male reproductive system, leading to compromised fertilizing capacity of sperm. This defect is rescued by superimposing deletion of cannabinoid receptor 1 (Cnr1). Retention of Faah^−/− sperm on the egg zona pellucida provides evidence that the capacity of sperm to penetrate the zona barrier is hampered by elevated anandamide levels. Collectively, the results show that aberrant endocannabinoid signaling via CNR1 impairs normal sperm function. Besides unveiling a new regulatory mechanism of sperm function, this study has clinical significance in male fertility.

Inflammatory processes are involved in the initiation and maintenance of labor, suggesting that Toll-like receptor (TLR) activity within gestation-associated tissues, such as the placenta, might contribute to the process of parturition. Expression of transcripts for TLR1–TLR10 was examined in term (>37 wk of gestation) human placentas collected in the absence of labor (elective caesarean sections; ECS; n = 11) and after the completion of labor (normal vaginal delivery; NVD; n = 12). Placental explants were cultured in the presence of agonists for TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9, and cytokine production after 24 h was examined. All placentas expressed transcripts for TLR1–TLR10. Reactivity to all agonists except CpG oligonucleotides was observed, indicating that, other than TLR9, all of the receptors studied yielded functional responses. Placental explants prepared from NVD placentas (n = 17) produced significantly more TNFA in response to lipopolysaccharide (TLR4 agonist) and resiquimod (TLR7/8 agonist) than explants from ECS placentas (n = 17). In contrast, gene expression analysis revealed that only transcripts for TLR2 and TLR5 were significantly elevated in association with labor. The human term placenta expresses a variety of functional TLRs, indicating that this family of receptors has an important role in parturition via as yet undetermined cell types and signaling pathways.

CD44 on macrophages is recognized as a phagocytic receptor involved in the phagocytosis of apoptotic cells. Recently, we detected CD44 on macrophages in atretic follicles during atresia. In this study, we evaluated the distribution of the principal CD44 ligand hyaluronan (HA) and the expressions of HA synthases (HAS: HAS1, HAS2, and HAS3) during atresia in pig ovaries. We determined the 2139-bp sequence of Sus scrofa HAS1 and raised an anti-HAS1 polyclonal antibody. The S. scrofa HAS1 sequence contained six putative HA-binding motifs and conserved amino acid residues crucial for GlcNac transferase activity. HAS1 mRNA expression was upregulated during atresia; however, HAS2 and HAS3 mRNA expression levels were low and very low to undetectable, respectively. Western blotting showed that HAS1 was markedly upregulated during atresia. Immunohistochemical analyses revealed HAS1 distribution in theca cells of healthy and early atretic (stages I and II) follicles and in progressing atretic (stage III) follicles. Hyaluronan was visualized with the HA-binding protein; it accumulated in the theca layer during all stages and in stage III follicles. Hyaluronan assay showed a significantly increased HA concentration in follicular fluid at stage III. Flow cytometry showed HAS1 expression in 55.7% of SIRPA-positive macrophages in stage III follicles. Our results suggest that the HA concentration in follicular fluids increased during atresia and that HAS1 may be the dominant HAS protein in theca cells to produce HA in pig ovaries.

The Hedgehog (Hh) signaling pathway affects fetal testis growth. Recently, we described the dynamic cellular production of Hh signaling pathway components in juvenile and adult rodent testes. The Hh signaling is understood to regulate cord formation in the fetal testis, but minimal knowledge exists regarding how Hh signaling impacts the postnatal testis. To investigate this, we employed hanging drop cultures, which are used routinely in embryoid body formation. This approach has the advantage of using small media volume, and we examined its suitability for short-term culture of both murine embryonic gonads and adult testis tubules. The effects of cyclopamine, a specific Hh signaling inhibitor, were examined following culture of Embryonic Day 11.5 urogenital ridges (as control) and adult seminiferous tubule fragments for 24–48 h using histological, cell proliferation, and gene expression analyses. Cultured embryonic testes displayed generally normal cord structure, anti-Müllerian hormone (Amh) expression, and cell proliferation; known Hh target gene expression (Gli1, osteopontin, official symbol Spp1, and Amh) was altered in response to cyclopamine. Cultured adult tubules exhibited some loss of seminiferous epithelium organization over 48 h. Spermatogonia continued to proliferate, however, and no significant loss of viability was noted overall. Addition of cyclopamine significantly affected levels of Gli1, Igfbp6, Ccnd2 (cyclin D2), Ccnb1 (cyclin B1), Spp1, Kit, and Amh mRNAs; these genes have been shown previously to be expressed in Sertoli and germ cells. These novel results identify Hh target genes in the testis and demonstrate this signaling pathway likely affects cell survival and differentiation in the context of normal adult testis.

In rat ovary chronic cold stress increases sympathetic nerve activity, modifies follicular development, and initiates a polycystic condition. To see whether there is a relationship between the previously described changes in follicular development and metabolic changes similar to those in women with polycystic ovary, we have studied the effect of chronic cold stress (4°C for 3 h/day, Monday to Friday, for 4 wk) on insulin sensitivity and the effect of insulin on sympathetic ovarian activity. Although cold-stressed rats ate more than the controls, they did not gain more weight. Insulin sensitivity, determined by hyperinsulinemic-euglycemic clamp, was significantly increased in the stressed animals. Insulin in vitro increased the basal release of norepinephrine from the ovaries of control rats but not from those of stressed rats, suggesting a local neural resistance to insulin in stressed rats. The levels of mRNA and protein for IRS1 and SLC2A4 (also known as GLUT4), molecules involved in insulin signaling, decreased significantly in the ovaries but not in the muscle of stressed rats. This decrease was preferentially located in theca-interstitial cells compared with granulosa cells, indicating that theca cells (the only cells directly innervated by sympathetic nerves) are responsible for the ovarian insulin resistance found in stressed rats. These findings suggest that ovarian insulin resistance produced by chronic stress could be in part responsible for the development of the polycystic condition induced by stress.

Endometriosis has been associated with a reduced response to progesterone in both the eutopic and ectopic endometrium. In this study we evaluated OVGP1 and steroid receptor expression in oviducts of baboons with endometriosis during the midsecretory phase and determined whether progesterone resistance associated with endometriosis also occurs in the oviduct. Oviducts obtained during the window of uterine receptivity (Day 10 postovulation [PO]) from animals with induced and spontaneous disease were compared to control animals during the proliferative stage and in the implantation window as well as animals treated with the progesterone receptor (PGR) antagonist ZK 137.299 (ZK). OVGP1 was significantly higher in animals with endometriosis compared with Day 10 PO controls and was similar to that seen in the late proliferative phase and in ZK-treated animals. Baboons with spontaneous endometriosis also showed a similar persistence of OVGP1, which was correlated with the maintenance of estrogen receptor 1 (ESR1) in the epithelial cells of animals with endometriosis. However, epithelial cell height and the percentage of ciliation were not affected by endometriosis. These data imply that the normal antagonism of progesterone on ESR and OVGP1, which results in their downregulation during the window of implantation, is absent in animals with endometriosis. This was confirmed further when the action of PGR was antagonized in animals without disease, which also resulted in the persistence of ESR1 and OVGP1. These studies suggest that an aberrant oviductal environment may be an additive factor that contributes to endometriosis-associated infertility.

Epididymal protease inhibitor (eppin [official symbol, SPINLW1]) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human semenogelin on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight-line distance, and decreased velocity. Anti-eppin treatment of spermatozoa significantly increased the amount of cAMP present in nonprogressive spermatozoa; however, approximately 25% of antibody-treated spermatozoa could be rescued by the addition of cAMP-acetoxymethyl ester, indicating that anti-eppin-treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human semenogelin has a concentration-dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block semenogelin binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to semenogelin. Addition of affinity-purified antibodies made to the dominant C-terminal epitope of eppin had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity, and straight distance). Our results suggest that the eppin-semenogelin binding site is critical for the removal of semenogelin in vivo during semen liquefaction and for the initiation of progressive motility. We conclude that the eppin-semenogelin binding site on the surface of human spermatozoa is an ideal target for a nonsteroidal male contraceptive.

A universal response to cellular stress is the expression of transformation-related protein 53 (TRP53). This transcription factor reduces cell proliferation and/or survival and is classed as a tumour suppressor protein. Several stresses (including culture) cause increased TRP53 expression in blastocysts and their reduced long-term developmental potential. This study shows that culture from the zygote stage (but not the 2-cell stage) reduced the development of C57BL6 inbred (but not hybrid) strain mouse embryos. Reduced viability was TRP53 dependent, being partially reversed by a TRP53 inhibitor (Pifithrin-alpha). However, the presence of culture did not cause an increase in Trp53 mRNA levels (levels were reduced following culture, P < 0.001). Transformed mouse 3T3 cell double minute 2 (MDM2) causes the ubiquitination and degradation of TRP53. MDM2 activation is accompanied by phosphorylation of Ser-166, and this is commonly catalyzed by the phosphatidylinositol-3 kinase and RAC-alpha serine/threonine-protein kinase (AKT) signaling pathway. Paf is an autocrine embryotrophin that activates the phosphatidylinositol-3 kinase/AKT pathway. High levels of TRP53 expression occurred following the culture of zygotes lacking the Paf receptor (Ptafr^−/−) and following inhibition of phosphatidylinositol-3 kinase or AKT. Inhibition of MDM2 caused a Trp53-dependent reduction in zygote development. Inbred strain embryos cultured from the zygote stage expressed less phosphorylated MDM2 than similar embryos collected from the uterus. The addition of Paf to the media caused increased phosphorylation of MDM2, and this was blocked by inhibitors of phosphatidylinositol-3 kinase and AKT. The study identifies trophic ligand signaling via the activation of phosphatidylinositol-3 kinase and AKT as a mechanism resulting in the activation of MDM2.

The nutrient requirements and metabolic pathways used by the developing embryo transition from predominantly pyruvate during early cleavage stages to glucose at the blastocyst; however, the complexities involved in the regulation of metabolism at different developmental stages are not clear. The aims of this study were to examine the role of the malate-aspartate shuttle (MAS) in nutrient metabolism pathways in the developing mouse blastocyst and the consequences of impaired metabolism on embryo viability and fetal and placental growth. Eight-cell-stage mouse embryos were cultured in the presence of the MAS inhibitor amino-oxyacetate, with or without pyruvate as an energy substrate in the media. When the MAS was inhibited, the rate of glycolysis and lactate production was significantly elevated and glucose uptake reduced, relative to control cultured embryos in the presence of pyruvate. Despite these changes in embryo metabolism, this did not influence development to the blastocyst stage, but it did reduce the number of inner cell mass and trophectoderm cells. When these embryos were transferred to psuedopregnant females, inhibition of the MAS significantly reduced the proportion of embryos that implanted and developed into fetuses on Day 18 of pregnancy. Finally, fetal growth was reduced while placental weight was maintained, leading to a decreased fetal:placental weight ratio relative to control embryos. These results suggest that impaired metabolism of glucose in the blastocyst via the MAS alters the ability of the embryos to implant and form a pregnancy and leads to reduced fetal weight, likely via altered placental development and function.

Successful pregnancy depends on the precise regulation of extravillous trophoblast (EVT) invasion into the uterine decidua, primarily by decidua-derived factors. In humans, during early pregnancy interleukin 11 (IL11) is maximally expressed in the decidua, with its receptor, IL11 receptor alpha (IL11RA), also identified on invasive EVTs in vivo. Although a role for IL11 in EVT migration has been established, whether it also plays a role in regulating EVT invasion is unknown. We investigated whether IL11 influences human EVT invasion and the signaling pathways and underlying mechanisms that may be involved, using the HTR-8/SVneo immortalized EVT cell line and primary EVTs as models for EVTs. Interleukin 11 (100 ng/ml) significantly inhibited invasion of EVT cells by 40% to 60% (P < 0.001). This effect was abolished by inhibitors of signal transducer and activator of transcription 3 (STAT3) but not of mitogen-activated protein kinase (MAPK) pathways. Interleukin 11 (100 ng/ml) had no effect on matrix metallopeptidases 2 and 9 (MMP2 and MMP9), tissue inhibitors of MMP (TIMP1, TIMP2, and TIMP3), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and serpin peptidase inhibitors 1 and 2 (SERPINE1 and SERPINE2) in EVT-conditioned media and/or cell lysates. Interleukin 11 (100 ng/ml) also did not regulate EVT cell adhesion or integrin expression. These data demonstrate that IL11 inhibits human EVT invasion via STAT3, indicating a likely role for IL11 in the decidual restraint of EVT invasion during normal pregnancy.

Sperm from the toad Bufo arenarum must penetrate the egg jelly before reaching the vitelline envelope (VE), where the acrosome reaction is triggered. When the jelly coat is removed, sperm still bind to the VE, but acrosomal exocytosis is not promoted. Our previous work demonstrated that diffusible substances of the jelly coat, termed “egg water” (EW), triggered capacitation-like changes in B. arenarum sperm, promoting the acquisition of a transient fertilizing capacity. In the present work, we correlated this fertilizing capacity with the ability of the sperm to undergo the acrosome reaction, further substantiating the role of the jelly coat in fertilization. When sperm were exposed to the VE, only those preincubated in EW for 5 or 8 min underwent an increase in the intracellular Ca² concentration ([Ca² ]_i), which led to acrosomal exocytosis. Responsiveness to the VE was not acquired on preincubation in EW for 2 or 15 min or in Ringer solution regardless of the preincubation time. In contrast, depletion of intracellular Ca² stores (induced by thapsigargin) promoted [Ca² ]_i rise and the acrosome reaction even in sperm that were not exposed to EW. Acrosomal exocytosis was blocked by the presence of Ca² chelators independent of whether a physiological or pharmacological stimulus was used. However, Ni² and mibefradil prevented [Ca² ]_i rise and the acrosome reaction of sperm exposed to the VE but not of sperm exposed to thapsigargin. These data suggest that the acrosomal responsiveness of B. arenarum sperm, present during a narrow period, is acquired during EW incubation and involves the modulation of a voltage-dependent Ca² channel.

Advances in treatment for testicular cancer that include the coadministration of bleomycin, etoposide, and cisplatin (BEP) have brought the cure rate to higher than 90%%. The goal of this study was to elucidate the impact of BEP treatment on gene expression in male germ cells. Brown-Norway rats were treated for 9 wk with vehicle (0×) or BEP at doses equivalent to 0.3× and 0.6× the human dose. At the end of treatment, spermatogenesis was affected, showing altered histology and a decreased sperm count; spermatozoa had a higher number of DNA breaks. After 9 wk of treatment, round spermatids were isolated, and RNA was extracted and probed on Rat230–2.0 Affymetrix arrays. Of the 31 099 probe sets present on the array, 59%% were expressed in control round spermatids. BEP treatment significantly altered the expression of 221 probe sets, with at least a 1.5-fold change compared with controls; 80%% were upregulated. We observed a dose-dependent increase in the expression of oxidative stress response genes and no change in the expression of genes involved in DNA repair. BEP upregulated genes were implicated in pathways related to Jun and Junb protooncogenes. Increased mRNA levels of Jun and Junb were confirmed by quantitative RT-PCR; furthermore, JUN protein was increased in elongating spermatids. Thus, BEP exposure triggers an oxidative stress response in round spermatids and induces many pathways that may lead to the survival of damaged cells and production of abnormal sperm.

Menopause is an important public health issue because of its association with a number of disorders. Androgens produced by residual ovarian tissue after menopause could impact the development of these disorders. It has been unclear, however, whether the postmenopausal ovary retains steroidogenic capacity. Thus, an ovary-intact mouse model for menopause that uses the occupational chemical 4-vinylcyclohexene diepoxide (VCD) was used to characterize the expression of steroidogenic genes in residual ovarian tissue of follicle-depleted mice. Female B6C3F1 mice (age, 28 days) were dosed daily for 20 days with either vehicle or VCD (160 mg kg⁻¹ day⁻¹) to induce ovarian failure. Ovaries were collected on Day 181 and analyzed for mRNA and protein. Acyclic aged mice were used as controls for natural ovarian senescence. Relative to cycling controls, expression of mRNA encoding steroidogenic acute regulatory protein (Star); cholesterol side-chain cleavage (Cyp11a1); 3beta-hydroxysteroid dehydrogenase (Hsd3b); 17alpha-hydroxylase (Cyp17a1); scavenger receptor class B, type 1 (Scarb1); low-density lipoprotein receptor (Ldlr); and luteinizing hormone receptor (Lhcgr) was enriched in VCD-treated ovaries. In acyclic aged ovaries, mRNA expression for only Cyp17a1 and Lhcgr was greater than that in controls. Compared to cycling controls, ovaries from VCD-treated and aged mice had similar levels of HSD3B, CYP17A1, and LHCGR protein. The pattern of protein immunofluorescence staining for HSD3B in follicle-depleted (VCD-treated) ovaries was homogeneous, whereas that for CYP17A1 was only seen in residual interstitial cells. Circulating levels of FSH and LH were increased, and androstenedione levels were detectable following follicle depletion in VCD-treated mice. These findings support the idea that residual ovarian tissue in VCD-treated mice retains androgenic capacity.

This study aims to analyze, in mice, the long-term effects of delayed fatherhood on postnatal development, spontaneous motor activity, and learning capacity of offspring. Hybrid parental-generation (F₀) males, at the age of 12, 70, 100, and 120 wk, were individually housed with a randomly selected 12-wk-old hybrid female. The resulting first-generation (F₁) offspring were tested for several developmental and behavioral variables. Cumulative percentage of F₁ pups that attained immediate righting in the 120-wk group was lower than that found in the 12-, 70-, and 100-wk groups. Furthermore, the postnatal day of attaining immediate righting was higher in pups from the 120-wk group when compared to pups from the other age-groups. At the age of 20 wk, F₁ offspring from the 120-wk group displayed lower counts of motor activity than offspring from the 12-, 70-, and 100-wk groups. One week later, a higher percentage of offspring from the 100- and 120-wk groups entered the dark compartment during the retention trial of the passive-avoidance test when compared to offspring from the 12-wk group. Offspring from the 120-wk group exhibited also lower step-through latency in the retention trial than offspring from the 12-, 70-, and 100-wk groups. These results show that advanced paternal age at conception has long-term effects on preweaning development, spontaneous motor activity, and reduced passive-avoidance learning capacity of mouse offspring.

This study aims to analyze, in mice, the long-term effects of delayed fatherhood on reproductive fitness and longevity of offspring. Hybrid parental-generation (F₀) males, at the age of 12, 70, 100, and 120 wk, were individually housed with a randomly selected 12-wk-old hybrid female. The reproductive fitness of first-generation (F₁) females was tested from the age of 25 wk until the end of their reproductive life. In F₁ males, the testing period ranged from the age of 52 wk until death. Breeding F₁ females from the 120-wk group displayed interbirth intervals longer than females from the 12-, 70-, and 100-wk groups. Furthermore, F₂ pups begotten by F₁ studs exhibited weaning weights lower than pups from the 12- and 70-wk groups. Offspring from the 120-wk group exhibited shorter survival times associated with lower incidence of tumorigenesis and higher loss of body weight when approaching death when compared to F₁ offspring from younger age-groups. The results indicate that advanced paternal age at conception has negative long-term effects on reproductive fitness and longevity of offspring in the mouse model.

Murine epididymal spermatozoa were dispersed in a medium of native osmolality and then transferred to a hypo-osmotic medium to mimic the physiological osmotic challenge, as encountered upon ejaculation into the female tract. The addition of quinine to block sperm K -channels for volume regulation resulted in a size increase of viable cells. Preincubation in 0.1 mM HgCl₂, a standard aquaporin inhibitor, prevented such cell swelling. Addition of the K -ionophore valinomycin to quinine-swollen sperm reversed the swelling, but not after pretreatment of the swollen sperm by HgCl₂. Aqp7, Aqp8, and Aqp9 mRNAs were identified in spermatozoa by RT-PCR, and the entire open reading frames were sequenced and compared with the GenBank database. Western blotting demonstrated specific protein signals for sperm AQP7 and AQP8 expression but probably not AQP9. The role of Hg² -insensitive AQP7, if any, in sperm volume regulation was studied in transgenic mice. Spermatozoa from Aqp7^−/− mice were the same size as wild-type sperm in basal conditions. Quinine-swollen volume, swelling reversal by valinomycin, and inhibition by Hg² were also similar, indicating efficient water transport in the absence of AQP7. However, both water influx and efflux occurred faster in Aqp7^−/− sperm than wild-type. This faster water movement in the knockout mouse spermatozoa was explainable by an upregulation of Aqp8 expression as revealed by quantitative PCR. Therefore, the Hg² -sensitive AQP8, which was localized in elongated spermatids and spermatozoa, is a likely candidate for a water channel responsible for physiological sperm volume regulation crucial to in vivo fertilization.

We identified HMGB4, a novel member of the HMGB family lacking the acidic tail typically found in this family. HMGB4 is strongly and preferentially expressed in the adult mouse testis and weakly in the brain, but not in many other tissues. HMGB4 associates with chromatin, and in transfection assays, in contrast to HMGB1, it acts as a potent transcriptional repressor. During spermatogenesis, HMGB4 is present in the euchromatin of late pachytene spermatocytes and haploid round spermatids, whereas stronger expression is observed during the elongation phase, where it localizes to the basal pole of the nucleus in a manner mutually exclusive with H1FNT (H1T2) localized at the apical pole. HMGB4 basal localization is lost in H1FNT-mutant spermatids, showing that H1FNT provides a positional cue for organizing chromatin domains within the nucleus. These results show that HMGB4 and H1FNT specify distinct chromatin domains at the apical and basal poles of the elongating spermatid nucleus.

As a key degrader of fibrillar collagens, matrix metalloproteinase 13 (MMP13), may contribute to the progression of pelvic organ prolapse. Here we aimed to define the regulation of MMP13 by estradiol and progesterone in the vaginal supportive tissues. Fibroblasts cultured from the arcus tendineous fasciae pelvis of three pre- and three postmenopausal women with prolapse were treated with 17-beta-estradiol (E2), progesterone (P4), E2 P4, or E2 ICI 182,780 (ICI). Collagenase inhibitor I (CI) and MG-132 were employed to investigate the mechanism of MMP13 degradation into inactive fragments (fragmentation) by hormones. The regulation of MMP13 in vivo was assessed by comparing tissues of ovariectomized (ovx) vs. sham-operated rats. Expression of MMP13 (proenzyme and active and fragment forms) was quantitated by Western immunoblotting, and MMP13 enzymatic activity was measured using a substrate degradation assay. The amount of cellular active MMP13 and MMP13 proteolytic activity decreased in the presence of hormones. The decrease was paralleled by increased proenzyme and fragment forms. MG-132, not CI, suppressed cellular MMP13 fragmentation. Active MMP13 increased in rats following ovx and was suppressed by E2 P4 supplementation. Active MMP13 is suppressed in vivo and in vitro by estradiol and progesterone, suggesting a protective effect against vaginal supportive tissue deterioration.

Prunella vulgaris (PV), a commonly used Chinese herb, also known as Self-heal, has a wide range of reported medicinal activities. By screening multiple herbs using the endometrial cancer cell line, ECC-1, and an alkaline phosphatase detection assay, we found that PV displayed significant antiestrogenic activity. We investigated the possible usefulness of antiestrogenic activity using both in vitro and in vivo models of endometrial function. Using the well-differentiated, hormone-responsive endometrial cell line, ECC-1, PV extract, at concentrations that were not toxic to the cells, significantly reduced alkaline phosphatase activity and cell proliferation in response to estrogen in a dose-dependent manner. The expression of CYR61, an estrogen-induced protein, was blocked in ECC-1 cells by both the antiestrogen ICI 182 780 and PV extract. Interestingly, PV extract did not appear to directly inhibit estrogen signaling. Rather, we found that its activities were probably related to an ability to function as an aryl hydrocarbon receptor (AHR) agonist in ECC-1 cells. In support of this hypothesis, we noted that PV induced CYP1A1, CYP1B1, and AHR repressor expression in a dose-dependent manner—responses that were blocked by small interfering RNA treatment to reduce AHR and specific AHR antagonists. Ovariectomized immunodeficient RAG-2/gamma(c) knockout mice implanted with human endometrial xenografts developed implants only when treated with estrogen. Mice treated with estrogen and PV tea in their drinking water had fewer and smaller xenograft implants compared with their estrogen-treated counterparts that drank only water (P < 0.05). Analysis of the resulting implants by immunohistochemistry demonstrated persistent estrogen receptor (ER), but reduced proliferation and CYR61 expression. Mouse uterine tissue weight in PV-treated mice was not different from controls, and cycle fecundity of intact C57 female mice was unaffected by PV tea treatment. PV, or Self-heal, exhibits significant antiestrogenic properties, both in vitro and in vivo. This activity is likely due to the ability of PV-activated AHR to interfere with estrogen. This herb may be useful as an adjunct for the treatment of estrogen-dependent processes like endometriosis and breast and uterine cancers. Full characterization of this herb will likely provide new insights into the crosstalk between AHR and ESR1, with potential for therapeutic applications in women.

Sirtuins (SIRTs) are class-III NAD-dependent histone deacetylases (HDACs) that regulate various physiological processes. Inactivation of SIRT1 in the mouse leads to male sterility, but the molecular mechanisms responsible for this phenotype have not been determined. Here we show that fetal testis development appears normal in Sirt1^−/− mice. In contrast, the first round of spermatogenesis arrests before the completion of meiosis with abundant apoptosis of pachytene spermatocytes, abnormal Leydig and Sertoli cell maturation, and strongly reduced intratesticular testosterone levels. We show that this phenotype is the consequence of diminished hypothalamic gonadotropin-releasing hormone expression and strongly reduced luteinizing hormone levels. Rather than having an intrinsic effect on male germ cells per se, our results show that SIRT1 regulates spermatogenesis at postnatal stages by controlling hypothalamus-pituitary gonadotropin (HPG) signaling. In addition to its well studied role in control of metabolism and energy homeostasis, our results thus reveal a novel and critical function of SIRT1 in controlling HPG signaling. This phenotype is more severe than those previously described using mice bred on different genetic backgrounds, and highlights the fact that SIRT1 function is strongly modified by other genetic loci.