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1.  Parturition and Recruitment of Macrophages in Cervix of Mice Lacking the Prostaglandin F Receptor1 
Biology of reproduction  2007;78(3):438-444.
Parturition does not occur in transgenic mice lacking the prostaglandin F receptor (Ptgfr−/−) because luteolysis is forestalled and progesterone production persists. Ovariectomy of pregnant Ptgfr−/− mice leads to a decline in circulating progesterone and delivery of live pups. The objective of the present study was to test the hypothesis that immigration of macrophages and increased innervation of the cervix of Ptgfr−/− mice was associated with ripening and parturition. The cervix of pregnant Ptgfr−/− mice was studied on Days 15–21 after breeding; additional groups were ovariectomized on Day 19 of pregnancy, and the cervix obtained on Day 20 of pregnancy before birth or the next day at about 24 h after birth. On Days 18–19 of pregnancy, macrophage numbers and nerve fiber density increased more than 3-fold compared with findings in nonpregnant or Day 15 or 21 pregnant Ptgfr−/− mice. The magnitude and time course of these changes were comparable to those found in wild-type controls that delivered on Day 19 after breeding. Thus, the mechanism regulating macrophage immigration, innervation, and cervical remodeling in Ptgfr−/− mice with delayed parturition is similar to wild-type controls that deliver at term. By contrast, ovariectomy forestalled the decrease in cervical macrophages in Ptgfr−/− mice. By Day 21 after breeding, macrophage numbers more than double those after ovariectomy, relative to those found in pregnant Ptgfr−/− mice, whereas nerve fiber density was the same regardless of birth. Density of collagen structure in these mice directly matched macrophage traffic in the cervix. The findings indicate that the prostaglandin F2alpha receptor and progesterone withdrawal are a necessary part of the final common pathway for ripening of the cervix and the process of parturition.
PMCID: PMC4237585  PMID: 18003949
cervix; cyclooxygenase; labor; leukocyte; macrophage; neuropeptides; parturition; progesterone; progesterone receptor
2.  Functional Redundancy of TGF-beta Family Type I Receptors and Receptor-Smads in Mediating Anti-Müllerian Hormone-Induced Müllerian Duct Regression in the Mouse1 
Biology of reproduction  2008;78(6):994-1001.
Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.
PMCID: PMC4023255  PMID: 18322278
AMH; AMHR2; female reproductive tract; male reproductive tract; Müllerian ducts; signal transduction; Wolffian duct
Biology of reproduction  2013;89(6):10.1095/biolreprod.113.113688.
Dynamic control of maternal blood flow to the placenta is critical for healthy pregnancy. In many tissues, microvasculature arteries control flow. The uterine/endometrial vascular bed changes during pregnancy include physiological remodeling of spiral arteries from constricted artery–like structures to dilated vein-like structures between gestation days (gd)8 and12 in mice and weeks 12–16 in humans. These changes occur, in part, due to local environmental changes such as decidualization, recruitment of maternal uterine natural killer cells and invasion of conceptus-derived trophoblasts. No current preparations permit in vivo testing of decidual microvascular reactivity. We report an in vivo intravital fluorescence microscopy model that permits functional study of the entire uterine microvascular bed (uterine, arcuate, radial, basal and spiral arteries) in gravid C57BL/6 mice. Vascular reactivities were measured at gd8 pre-spiral arterial remodeling and gd12 (post-remodeling) to a range of concentrations of adenosine (ADO, 10−8–10−6M), acetylcholine (ACh, 10−7–10−5M), phenylephrine (PHE, 10−7–10−5M) and angiotensin II (AngII, 10−8–10−6M). At baseline, each arterial branch order was significantly more dilated on gd12 than gd8. Each microvascular level responded to each agonist on gd8 and gd12. At gd12, vasodilation to ADO was attenuated in uterine, arcuate and basal arteries while constrictor activity to AII was enhanced in uterine and arcuate arteries. The tendency for increasing vasoconstriction between gd8 to gd12 and the constrictor responses of modified spiral arteries were unexpected findings that may reflect influences of the intact in vivo environment rather than inherent properties of the vessels and be relevant to ongoing human pregnancies.
PMCID: PMC3866111  PMID: 24174571 CAMSID: cams3647
decidua; pregnancy; blood flow; artery; vascular remodeling
4.  Endometrial Receptivity Defects and Impaired Implantation in Diabetic NOD Mice1 
Biology of reproduction  2012;87(2):30.
Implantation failure is a major hurdle to a successful pregnancy. The high rate of postimplantation fetal loss in nonobese diabetic (NOD) mice is believed to be related to an abnormal decidual production of interferon (IFN)gamma. To address whether diabetes alters the natural events associated with successful implantation, certain morphological and molecular features of uterine receptivity in diabetic NOD (dNOD) mice were examined in normally mated pregnancy and in concanavalin A (ConA)-induced pseudopregnancy. As opposed to normoglycemic NOD (cNOD) mice, dNOD mice expressed retarded maturation of their uterine pinopodes and overexpressed MUC1 mucin at implantation sites (P < 0.001). Uterine production of leukemia inhibitory factor (LIF) and phosphorylation of uterine NFkappaBp65 and STAT3-Ty705 were found to be low (P < 0.01) during Day 4.5 postcoitum, whereas IFNgamma was aberrantly overexpressed. Loss of temporal regulation of progesterone receptor A (PR A) and PR B, together with aberrantly increased expression of the protein inhibitor of activated STAT-y (PIASy) (P < 0.01) and reduced recruitment (P < 0.01) of the latter to nuclear progesterone receptor sites were prominent features of decidualization failure occurring at peri-implantation in dNOD mice. In conclusion, the aberrant expression of endometrial IFNgamma in dNOD mice is associated with a nonreceptive endometrial milieu contributing to peri-implantation embryo loss in type 1 diabetes.
PMCID: PMC3726408  PMID: 22539679 CAMSID: cams3210
IFNγ; implantation failure in type 1 diabetes; MUC1; NFκB; NOD mice; PIASy; progesterone receptor; STAT3; uterodomes
5.  Analysis of Maternal and Fetal Cardiovascular Systems During Hyperglycemic Pregnancy in the Non-Obese Diabetic Mouse 
Biology of reproduction  2013;88(6):151.
Pre-conception or gestationally-induced diabetes increases morbidities and elevates long-term cardiovascular disease risks in women and their children. Spontaneously hyperglycemic (d)-NOD/ShiLtJ females, a type 1 diabetes model, develop bradycardia and hypotension after midpregnancy compared with normoglycemic, age and gestation day (gd)-matched controls (c-NOD). We hypothesized that onset of the placental circulation at gd9–10 and rapid fetal growth from gd14 correlate with aberrant hemodynamic outcomes in d-NOD females. To develop further gestational time course correlations between maternal cardiac and renal parameters, high-frequency ultrasonography was applied to virgin and gd8–16 d- and c-NODs. Cardiac output and left ventricular (LV) mass increased in c- but not d-NODs. Ultrasound and postmortem histopathology showed overall greater LV dilation in d- than c-NOD mice in mid-late gestation. These changes suggest blunted remodeling and altered functional adaptation of d-NOD hearts. Umbilical cord ultrasounds revealed lower fetal heart rates from gd12 and lower umbilical flow velocities at gd14 and 16 in d- versus c-NOD pregnancies. From gd14–16, d-NOD fetal losses exceeded those of c-NOD. Similar aberrant responses in human diabetic pregnancies may elevate postpartum maternal and child cardiovascular risk, particularly if mothers lack adequate prenatal care or have poor glycemic control over gestation.
PMCID: PMC3699463  PMID: 23636813 CAMSID: cams2944
Pregnancy; diabetes; NOD mouse; cardiovascular system; cardiac adaptations
6.  Survival and size are differentially regulated by placental and fetal PKBα/Akt1 
Biology of reproduction  2010;84(3):537-545.
The importance of the placental circulation is exemplified by the correlation of placental size and blood flow with fetal weight and survival during normal and compromised human pregnancies in conditions as preeclampsia and intrauterine growth restriction (IUGR). Utilizing non-invasive magnetic resonance imaging (MRI) we evaluated the role of PKBα/Akt1, a major mediator of angiogenesis, on placental vascular function. PKBα/Akt1 deficiency reduced maternal blood volume fraction without affecting the integrity of the feto-maternal blood barrier. In addition to angiogenesis, PKBα/Akt1 regulates additional processes related to survival and growth. In accordance to reports in adult mice, we demonstrated a role for PKBα/Akt1 in regulating chondrocyte organization in fetal long bones. Using tetraploid complementation experiments, with PKBα/Akt1-expressing placentas, we found that while placental PKBα/Akt1 restored fetal survival, fetal PKBα/Akt1 regulated fetal size, as tetraploid complementation did not prevent intrauterine growth retardation. Histological examination of rescued fetuses showed reduced liver blood vessel and renal glomeruli capillary density in PKBα/Akt1 null fetuses, both of which were restored by tetraploid complementation. However, bone development was still impaired in tetraploid-rescued PKBα/Akt1 null fetuses. Although PKBα/Akt1-expressing placentas restored chondrocyte cell number in the hypertrophic layer of humeri, fetal PKBα/Akt1 was found to be necessary for chondrocyte columnar organization. Remarkably, a dose-dependent phenotype was exhibited for PKBα/Akt1, when examining PKBα/Akt1 heterozygous fetuses as well as those complimented by tetraploid placentas. The differential role of PKBα/Akt1 on mouse fetal survival and growth may shed light on its roles in human IUGR.
PMCID: PMC3558738  PMID: 20980686
PKBα; placenta; fetus; tetraploid; MRI
7.  DBA-Lectin Reactivity Defines Mouse Uterine Natural Killer Cell Subsets with Biased Gene Expression 1 
Biology of reproduction  2012;87(4):81.
Endometrial decidualization, a process essential for blastocyst implantation in species with hemochorial placentation, is accompanied by an enormous but transient influx of Natural Killer (NK) cells. Mouse uterine (u)NK cell subsets have been defined by diameter and cytoplasmic granule number, reflecting stage of maturity and by histochemical reactivity with Periodic Acid Schiff’s (PAS) reagent, with or without co-reactivity with Dolichos biflorus agglutinin (DBA) lectin. We asked whether DBA− and DBA+ mouse uNK cells were equivalent using quantitative (q)RT-PCR analyses of flow separated, midpregnancy (gestation day (gd)10) cells and using immunohistochemistry. CD3E (CD3)-IL2RB (CD122)+DBA− cells were identified as the dominant Ifng transcript source. Skewed IFNG production by uNK cell subsets was confirmed by analysis of uNK cells from eYFP-tagged IFNG-reporter mice. In contrast, CD3E-IL2RB+DBA+ uNK cells expressed genes compatible with significantly greater potential for IL22 synthesis, angiogenesis and participation in regulation mediated by the renin-angiotensin system (RAS). CD3E-IL2RB+DBA+ cells were further divided into VEGFA+ and VEGFA− subsets. CD3E-IL2RB+DBA+ uNK cells but not CD3E-IL2RB+DBA− uNK cells arose from circulating, bone marrow-derived progenitor cells by gd6. These findings indicate the heterogeneous nature of mouse uNK cells and suggest that studies using only DBA + uNK cells will give biased data that does not fully represent the uNK cell population.
PMCID: PMC3467293  PMID: 22875907 CAMSID: cams2292
Cytokines; Decidua; Immunology; Pregnancy; Uterus
8.  Profiling Gene Expression During the Differentiation and Development of the Murine Embryonic Gonad1 
Biology of reproduction  2004;72(2):492-501.
The application of microarray technology to the study of mammalian organogenesis can provide greater insights into the steps necessary to elicit a functionally competent tissue. To this end, a temporal profile of gene expression was generated with the purpose of identifying changes in gene expression occurring within the developing male and female embryonic gonad. Gonad tissue was collected from mouse embryos at 11.5, 12.5, 14.5, 16.5, and 18.5 days postcoitum (dpc) and relative steady-state levels of mRNA were determined using the Affymetrix MGU74v2 microarray platform. Statistical analysis produced 3693 transcripts exhibiting differential expression during male and/or female gonad development. At 11.5 dpc, the gonad is morphologically indifferent, but at 12.5 dpc, transitions to a male or female phenotype are discernible by the appearance of testicular cords. A number of genes are expressed during this period and many share similar expression profiles in both sexes. As expected, the expression of two well-known sex determination genes, specifically Sry and Sox9, is unique to the testis. Beyond 12.5 dpc, differential gene expression becomes increasingly evident as the male and female tissue morphologically and physiologically diverges. This is evident by two unique waves of transcriptional activity occurring after 14.5 dpc in the male and female. With this study, a large number of transcripts comprising the murine transcriptome can be examined throughout male and female embryonic gonad development and allow for a more complete description of gonad differentiation and development.
PMCID: PMC3217241  PMID: 15496517
developmental biology; embryo; gene regulation; ovary; testis
9.  Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) and Maturation of Murine Gonocytes and Spermatogonia Induced by Retinoic Acid In Vitro1 
Biology of reproduction  2007;78(3):537-545.
Vitamin A deficiency in the mouse results in an arrest in the progression of undifferentiated spermatogonia to differentiating spermatogonia. The supplement of retinol to vitamin-A-deficient mice reinitiates spermatogenesis in a synchronous manner throughout the testes. It is unclear whether the effects of retinoids are the result of a direct action on germ cells or are indirectly mediated through Sertoli cells. The expression of Stimulated by retinoic acid gene 8 (Stra8), which is required for spermatogenesis, is directly related to the availability of retinoic acid (RA). Analysis of gene expression by microarrays revealed moderate levels of Stra8 transcript in gonocytes and high levels in A and B spermatogonia. Stra8 mRNA levels were greatly reduced or absent in germ cells once they entered meiosis. This study examined the effect of retinoic acid on cultured neonatal testes and isolated gonocytes/spermatogonia in vitro. THY1+ and KIT+ germ cells were isolated by magnetic-activated cell sorting from the testes of mice of different ages. Isolated germ cells were cultured and treated with either vehicle (ethanol) or RA without feeder cells. We found that 1) Stra8 is predominantly expressed in premeiotic germ cells, 2) RA stimulates gonocyte DNA replication and differentiation in cultured neonatal testes, 3) in the absence of feeder cells, RA directly induces the transition of undifferentiated spermatogonia to differentiating spermatogonia by stimulating Stra8 and Kit gene expression, 4) RA dramatically stimulates Stra8 expression in undifferentiated spermatogonia but has a lesser impact in differentiating spermatogonia, 5) endogenous Stra8 gene expression is higher in differentiating spermatogonia than in undifferentiated spermatogonia and could mediate the RA effects on spermatogonial maturation, and 6) RA stimulates a group of genes involved in the metabolism, storage, transport, and signaling of retinoids.
PMCID: PMC3208258  PMID: 18032419
differentiation; gonocytes; in vitro; retinoic acid; spermatogenesis; spermatogonia; spermatogonial differentiation; Stra8
10.  Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) in Spermatogenic Cells Induced by Retinoic Acid: An In Vivo Study in Vitamin A-Sufficient Postnatal Murine Testes1 
Biology of reproduction  2008;79(1):35-42.
Vitamin A is required for male fertility and normal spermatogenesis. Retinoic acid (RA), an active metabolite of vitamin A, is necessary for spermatogonial maturation and proper entry of germ cells into meiotic prophase in the postnatal testes. The expression of Stra8, which is essential for successful meiosis in both male and female gonads and normal spermatogenesis, is directly related to the availability of RA. This study examined the developmental expression pattern of Stra8 transcript in both male and female gonads, provided specific cellular localization of STRA8 protein in the postnatal and adult testis, and investigated RA actions in adult germ cells in a vitamin A-sufficient condition. The peak of Stra8 mRNA expression coincided with the onset of meiosis in postnatal testes. STRA8 protein was detected in gonocytes as early as 5 days postpartum. The expression of STRA8 protein in the neonatal testes was not uniform among spermatogonia, perhaps heralding the asynchronous beginning of spermatogenesis. In adult testes, the highest level of Stra8 mRNA and protein was found in seminiferous epithelial stages VI–VIII. STRA8 protein was localized to some type A and B spermatogonia, preleptotene spermatocytes, and early leptotene spermatocytes. In the vitamin A-sufficient adult testes, RA but not retinol acetate stimulated Stra8 mRNA expression. STRA8 protein expression in adult spermatogonia was induced by RA stimulation, suggesting its role in spermatogonial differentiation. Retinoic acid also increased the number of preleptotene spermatocytes exhibiting 5-bromo-2-deoxyuridine incorporation, indicating a more synchronized premeiotic DNA replication.
PMCID: PMC3208264  PMID: 18322276
in vivo; preleptotene spermatocytes; retinoic acid; spermatogonia; STRA8
11.  Kinesin Light-Chain KLC3 Expression in Testis Is Restricted to Spermatids1 
Biology of reproduction  2001;64(5):1320-1330.
Kinesins are tetrameric motor molecules, consisting of two kinesin heavy chains (KHCs) and two kinesin light chains (KLCs) that are involved in transport of cargo along microtubules. The function of the light chain may be in cargo binding and regulation of kinesin activity. In the mouse, two KLC genes, KLC1 and KLC2, had been identified. KLC1 plays a role in neuronal transport, and KLC2 appears to be more widely expressed. We report the cloning from a testicular cDNA expression library of a mammalian light chain, KLC3. The KLC3 gene is located in close proximity to the ERCC2 gene. KLC3 can be classified as a genuine light chain: it interacts in vitro with the KHC, the interaction is mediated by a conserved heptade repeat sequence, and it associates in vitro with microtubules. In mouse and rat testis, KLC3 protein expression is restricted to round and elongating spermatids, and KLC3 is present in sperm tails. In contrast, KLC1 and KLC2 can only be detected before meiosis in testis. Interestingly, the expression profiles of the three known KHCs and KLC3 differ significantly: Kif5a and Kif5b are not expressed after meiosis, and Kif5c is expressed at an extremely low level in spermatids but is not detectable in sperm tails. Our characterization of the KLC3 gene suggests that it carries out a unique and specialized role in spermatids.
PMCID: PMC3161965  PMID: 11319135 CAMSID: cams1886
gene regulation; meiosis; spermatid; spermatogenesis; testis
12.  Tsga10 Encodes a 65-Kilodalton Protein That Is Processed to the 27-Kilodalton Fibrous Sheath Protein1 
Biology of reproduction  2003;70(3):608-615.
We had previously reported the isolation of the testis-specific human gene Tsga10, which is not expressed in testes from two infertile patients. To study its role and function, we cloned the mouse homologue Mtsga10. Mtsga10 localizes to mouse chromosome 1, band B. This region is syntenic with human chromosome 2q11.2, where Tsga10 is located. We demonstrate that Mtsga10 mRNA is expressed in testis, but not in other adult tissues, and in several human fetal tissues and primary tumors. We uncovered that different species use different first exons and, consequently, different promoters. Using several antibodies, we discovered that, in mouse testis, Mtsga10 encodes a 65-kDa spermatid protein that appears to be processed to a 27-kDa protein of the fibrous sheath, a major sperm tail structure, in mature spermatozoa. Mtsga10 protein contains a putative myosin/Ezrin/radixin/moesin (ERM) domain. Transfection of fibroblasts with GFP-Mtsga10 fusion protein results in formation of short, thick filaments and deletion of the myosin/ERM domain abolished filament formation. Our results suggest the possibility that Tsga10 plays a role in the sperm tail fibrous sheath.
PMCID: PMC3158800  PMID: 14585816 CAMSID: cams1890
developmental biology; spermatid; spermatogenesis; testis
13.  Novel RING Finger Protein OIP1 Binds to Conserved Amino Acid Repeats in Sperm Tail Protein ODF11 
Biology of reproduction  2003;68(2):543-552.
Outer dense fibers (ODFs) and the fibrous sheath (FS) are unique structures of the mammalian sperm tail. Recently, progress has been made in the molecular cloning of ODF and FS proteins, and because of this, questions addressing the morphogenesis and underlying protein network that make up sperm tail structures and their function can now be addressed. Using the N-terminal leucine zipper motif of the major ODF protein ODF1, we had previously isolated interacting proteins Odf2, Spag4, and Spag5. We report here a yeast two-hybrid strategy to isolate a novel rat testicular protein, OIP1, that binds to the evolutionarily conserved Cys-Gly-Pro repeats in the C-terminus of ODF1. OIP1 is expressed in round spermatids as well as in spermatocytes and several somatic tissues, albeit at a lower level. No expression was detectable in epididymis, heart, and smooth muscle. OIP1 protein localizes to the sperm tail in a pattern expected for an ODF1-interacting protein. OIP1 belongs to the family of RING finger proteins of the H2 subclass. Deletion of the putative RING motif significantly decreased binding to ODF1. Genomic analysis of rat Oip1 and Oip1 homologs indicates that Oip1 is highly conserved. Oip1 is subject to differential splicing and alternative polyadenylation events. It is interesting that Oip1 mRNAs have been reported that lack the exon encoding the putative RING finger.
PMCID: PMC3158802  PMID: 12533418 CAMSID: cams1885
developmental biology; gamete biology; gametogenesis; spermatogenesis; testis
14.  A Novel Testicular RhoGAP-Domain Protein Induces Apoptosis1 
Biology of reproduction  2004;71(6):1980-1990.
The GTPase-activating proteins (GAPs) accelerate the hydrolysis of GTP to GDP by small GTPases. The GTPases play diverse roles in many cellular processes, including proliferation, cell motility, endocytosis, nuclear import/export, and nuclear membrane formation. Little is known about GAP-domain proteins in spermatogenesis. We isolated a novel RhoGAP domain-containing tGAP1 protein from male germ cells that exhibits unusual properties. The tGAP1 is expressed at low levels in early spermatogonia. Robust transcription initiates in midpachytene spermatocytes and continues after meiosis. The 175-kDa tGAP1 protein localizes to the cytoplasm of spermatocytes and to the cytoplasm and nucleus in spermatids. The protein contains four GAP domain-related sequences, in contrast to all other GAP proteins that harbor one such domain. No activity toward RhoA, Rac1, or Cdc42 could be detected. Results of transfection studies in various somatic cells indicated that low-level tGAP1 expression significantly slows down the cell cycle. Expression of higher levels of tGAP1 by infection of somatic cells with recombinant adenoviruses demonstrated that tGAP1 efficiently induces apoptosis, which to our knowledge is the first such demonstration for a RhoGAP protein. Based on its subcellular location in spermatids and its activity, tGAP1 may play a role in nuclear import/export.
PMCID: PMC3158803  PMID: 15306557 CAMSID: cams1891
apoptosis; spermatid; spermatogenesis; testis
15.  Spermatogenesis in Bclw-Deficient Mice1 
Biology of reproduction  2001;65(1):318-332.
Bclw is a death-protecting member of the Bcl2 family of apoptosis-regulating proteins. Mice that are mutant for Bclw display progressive and nearly complete testicular degeneration. We performed a morphometric evaluation of testicular histopathology in Bclw-deficient male mice between 9 days postnatal (p9) through 1 yr of age. Germ cell loss began by p22, with only few germ cells remaining beyond 7 mo of age. A complete block to elongated spermatid development at step 13 occurred during the first wave of spermatogenesis, whereas other types of germ cells were lost sporadically. Depletion of Sertoli cells commenced between p20 and p23 and continued until 1 yr of age, when few, if any, Sertoli cells remained. Mitochondria appeared to be swollen and the cytoplasm dense by electron microscopy, but degenerating Bclw-deficient Sertoli cells failed to display classical features of apoptosis, such as chromatin condensation and nuclear fragmentation. Macrophages entered seminiferous tubules and formed foreign-body giant cells that engulfed and phagocytosed the degenerated Sertoli cells. Leydig cell hyperplasia was evident between 3 and 5 mo of age. However, beginning at 7 mo of age, Leydig cells underwent apoptosis, with dead cells being phagocytosed by macrophages. The aforementioned cell losses culminated in a testis-containing vasculature, inter-tubular phagocytic cells, and peritubular cell “ghosts.” An RNA in situ hybridization study indicates that Bclw is expressed in Sertoli cells in the adult mouse testis. Consequently, the diploid germ cell death may be an indirect effect of defective Sertoli cell function. Western analysis was used to confirm that Bclw is not expressed in spermatids; thus, loss of this cell type most likely results from defective Sertoli cell function. Because Bclw does not appear to be expressed in Leydig cells, loss of Leydig cells in Bclw-deficient mice may result from depletion of Sertoli cells. Bclw-deficient mice serve as a unique model to study homeostasis of cell populations in the testis.
PMCID: PMC3049812  PMID: 11420255
aging; apoptosis; developmental biology; gametogenesis; Leydig cells; Sertoli cells; testes
16.  Matrix Metalloproteinase 9 (MMP9) Expression in Preeclamptic Decidua and MMP9 Induction by Tumor Necrosis Factor Alpha and Interleukin 1 Beta in Human First Trimester Decidual Cells1 
Biology of reproduction  2008;78(6):1064-1072.
Extravillous trophoblasts (EVTs) invade human decidua via sequential integrin-mediated binding and proteolysis of basement membrane proteins in the extracellular matrix (ECM). In preeclampsia, shallow EVT invasion impairs spiral artery and arteriole remodeling to reduce uteroplacental blood flow. Excess decidual cell-expressed matrix metalloproteinases (MMPs) 2 and 9, in response to preeclampsia-related interleukin 1 beta (IL1B) and tumor necrosis factor alpha (TNF), may inappropriately degrade these basement membrane proteins and impede EVT invasion. This study found significantly higher immunohistochemical MMP9 levels in decidual cells and adjacent interstitial trophoblasts in placental sections of preeclamptic versus gestational age-matched control women. In contrast, immunostaining for MMP2 and tissue inhibitor of matrix metalloproteinases 1 and 2 (TIMP1 and TIMP2) were similar in preeclamptic and control groups. First-trimester decidual cells were incubated with estradiol (E2) or E2 + medroxyprogesterone acetate (MPA), with or without TNF or IL1B. As measured by ELISA, both cytokines elicited concentration-dependent increases in secreted MMP9 levels that were unaffected by MPA. In contrast, secreted levels of MMP2, TIMP1, and TIMP2 were unchanged in all treatment groups. Substrate gel zymography and Western blotting confirmed that each cytokine increased secreted levels of MMP9 but not MMP2. Similarly, quantitative RT-PCR found that TNF and IL1B enhanced MMP9, but not MMP2, mRNA levels. At the implantation site, inflammatory cytokine-enhanced MMP9 may promote preeclampsia by disrupting the decidual ECM to interfere with normal stepwise EVT invasion.
PMCID: PMC3045968  PMID: 18276934
cytokines; decidua; extracellular matrix; matrix metalloproteinase; pregnancy
17.  WNT2 Regulates Proliferation of Mouse Granulosa Cells through Beta-Catenin1 
Biology of reproduction  2010;82(5):865-875.
WNTs are secreted extracellular signaling molecules that transduce their signals by binding to G protein-coupled receptors of the frizzled (FZD) family. They control diverse developmental processes such as cell fate specification, cell proliferation, cell differentiation, and apoptosis. Although WNT signaling has been shown to be essential for development of the ovary, its mechanistic role in folliculogenesis within the adult ovary has not been studied extensively. Therefore, the objective of this study was to investigate the regulation and function of WNT2 signaling in mouse granulosa cells. Immunostaining identified WNT2 as being expressed in granulosa cells throughout folliculogenesis, but with varying signal strength: in sequential sections, WNT2 immunoreactivity was strongest in healthy antral follicles but weak in atretic follicles. Knockdown of WNT2 expression using transfected siRNA decreased the proliferation rate of granulosa cells whereas WNT2 overexpression using a recombinant viral vector enhanced their proliferation. WNT2 knockdown led to accumulation of glycogen synthase kinase-3β (GSK-3β) in the cytoplasm but reduced the expression of β-catenin. Conversely, WNT2 overexpression reduced the expression of GSK-3β in the cytoplasm and induced β-catenin translocation from the membrane into the nucleus. β-catenin knockdown also inhibited the proliferation of granulosa cells, and neutralized the proliferation effect of WNT2 overexpression. WNT2/β-catenin signaling had a slight effect on the apoptosis of granulosa cells. Taken together, the data indicate that WNT2 regulates β-catenin localization in granulosa cells and WNT2/β-catenin signaling contributes to regulating their proliferation.
PMCID: PMC3025002  PMID: 20107203 CAMSID: cams1668
WNT; FZD; GSK-3β; β-catenin; AXIN; proliferation; granulosa cell
18.  The interactions between the stimulatory effect of Follicle-Stimulating Hormone and the inhibitory effect of estrogen on mouse primordial folliculogenesis 
Biology of reproduction  2009;82(1):13-22.
The murine primordial follicle pool develops largely within 3 days after birth through germline nest breakdown and enclosure of oocytes within pre-granulosa cells. The mechanisms that trigger primordial follicle formation are likely influenced by a transition from the maternal to fetal hormonal milieu at the time of birth. High levels of maternal estrogen maintain intact germline nest in fetal ovary and decrease of estrogen after birth is permissive to follicle formation. In the present study, we measured an increase in neonatal serum follicle-stimulating hormone (FSH), which corresponded to falling estradiol (E2) levels during the critical window of primordial follicle formation (postnatal days 1-3). To determine whether fetal hormones contribute in an active manner to primordial follicle formation, 17.5 days post coitus (dpc) mouse fetal ovaries were cultured in vitro in two concentrations of E2 (levels meant to reflect maternal and fetal levels of E2) and FSH for six days. High levels of E2 (10-6 M) inhibited germline nest breakdown and this effect was significantly reduced when fetal ovaries were cultured in low E2 concentration (10-10 M). FSH facilitated germline nest breakdown and primordial follicle formation under both high and low E2 culture conditions. Low E2 was identified as being more permissive for the effects of FSH on primordial follicle formation by stimulating the up-regulation of Fshr and activin betaA subunit expression, pre-granulosa cell proliferation, and oocyte survival. The decrease of E2 plus FSH after birth are critical for primordial follicle formation and the expression of oocyte-specific transcription factors (Figla and Nobox), in that inappropriate expose to the FSH or E2 during follicle formation resulted in premature or delayed primordial folliculogenesis. In conclusion, with the drop of E2 level after birth, FSH promotes primordial follicle formation in mice by stimulating local activin signaling pathways and the expression of oocyte-specific transcription factors.
PMCID: PMC2796699  PMID: 19641178
Follicle-stimulating hormone; Activin; Ovary; Primordial follicle
19.  Regulation of Lhb and Egr1 Gene Expression by GNRH Pulses in Rat Pituitaries Is Both c-Jun N-Terminal Kinase (JNK)- and Extracellular Signal-Regulated Kinase (ERK)-Dependent 
Biology of reproduction  2009;81(6):1206-1215.
Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1, and its two co-repressors Ngfi-A binding proteins 1 and 2 (NAB1 and NAB2), both in vivo and in cultured rat pituitary cells. In rats, fast GNRH pulses (every 30min) induced Egr1 primary transcript (PT) and mRNA stably 2-fold (p<0.05) for 1–24h. In contrast slow GNRH pulses (every 240min) increased Egr1 PT at 24h (6-fold; p<0.05), but increased Egr1 mRNA 4–5 fold between 4 and 24h. Both GNRH pulse frequencies increased EGR1 protein 3–4 fold. In cultured rat pituitary cells, GNRH pulses (every 60min) increased Egr1 (PT = 2.5–3 fold; mRNA = 1.5–2 fold; p<0.05). GNRH pulses had little effect on Nab1/2 PT/mRNAs either in vivo or in vitro. We also examined specific intracellular signaling cascades activated by GNRH. Inhibitors of Mitogen Activated Protein Kinases 8/9 (MAPK8/9 [aka JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [aka MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (aka p38) inhibitor SB203580 did not. In summary, pulsatile GNRH stimulates Egr1 gene expression and protein in vivo, but not in a frequency dependent manner. Additionally, GNRH induced Egr1 gene expression is mediated by MAPK8/9 and MAPK1/3, and both are critical for Lhb gene transcription.
PMCID: PMC2788048  PMID: 19710510
Gonadotropins; Egr1; MAPK8/9(JNK); MAPK1/3(ERK)
20.  Significant improvement in cloning efficiency of an inbred miniature pig by Scriptaid treatment after somatic cell nuclear transfer 
Biology of reproduction  2009;81(3):525-530.
The NIH miniature pig was developed specifically for xenotransplantation and has been extensively used as a large animal model in many other biomedical experiments. However the cloning efficiency of this pig is very low (less than 0.2%) and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Here we report that treatment with 500 nM Scriptaid, a novel HDACi, significantly enhanced the development SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) (21% vs. 9%, P < 0.05) were used as donors compared to the untreated group. Scriptaid treatment resulted in 8 pregnancies from 10 embryo transfers (ET) and 14 healthy NIH miniature pigs from 8 litters while no viable piglets (only 3 mummies) were obtained from 9 ETs in the untreated group. Thus Scriptaid dramatically increased the cloning efficiency when using inbred genetics from zero to 1.3%. In contrast, Scriptaid treatment decreased the blastocyst rate in IVF embryos (from 37% to 26%, P < 0.05). In conclusion, the extreme low cloning efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.
PMCID: PMC2731980  PMID: 19386991
SCNT; Histone acetylation; Scriptaid; inbred
21.  Overexpression of SK3 Channels Dampens Uterine Contractility to Prevent Preterm Labor in Mice 
Biology of reproduction  2008;78(6):1058-1063.
The mechanisms that control the timing of labor have yet to be fully characterized. In a previous study, the overexpression of small conductance calcium-activated K+ channel isoform 3 in transgenic mice, Kcnn3tm1Jpad/Kcnn3tm1Jpad (also known as SK3T/T), led to compromised parturition, which indicates that KCNN3 (also known as SK3) plays an important role in the delivery process. Based on these findings, we hypothesized that SK3 channel expression must be downregulated late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. Thus, we investigated the effects of SK3 channel expression on gestation and parturition, comparing SK3T/T mice to wild type (WT) mice. Here, we show in WT mice that SK3 transcript and protein are significantly reduced during pregnancy. We also found the force produced by uterine strips from Pregnancy Day 19 (P19) SK3T/T mice was significantly less than that measured in WT or SK3 knockout control (SK3DOX) uterine strips, and this effect was reversed by application of the SK3 channel inhibitor apamin. Moreover, two treatments that induce labor in mice failed to result in complete delivery in SK3T/T mice within 48 h after injection. Thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when SK3 channel activity is in excess. Our data indicate that SK3 channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice.
PMCID: PMC2930016  PMID: 18305226
ion channels; myometrium; parturition; pregnancy; preterm labor; SK3 channels; smooth muscle; uterus
22.  Estrogen Promotes Germ Cell and Seminiferous Tubule Development in the Baboon Fetal Testis1 
Biology of reproduction  2009;81(2):406-414.
The foundation for development of the male reproduction system occurs in utero, but relatively little is known about the regulation of primate fetal testis maturation. Our laboratories have shown that estrogen regulates key aspects of the physiology of pregnancy and fetal development. Therefore, in the present study, we characterized and quantified germ cells and Sertoli cells in the fetal baboon testis in late normal gestation (i.e., Day 165; term is 184 days) and in baboons administered the aromatase inhibitor letrozole throughout the second half of gestation to assess the impact of endogenous estrogen on fetal testis development. In untreated baboons, the seminiferous cords were comprised of undifferentiated (i.e., type A) spermatogonia classified by their morphology as dark (Ad) or pale (Ap), gonocytes (precursors of type A spermatogonia), unidentified cells (UI), and Sertoli cells. In letrozole-treated baboons, serum estradiol levels were decreased by 95%. The number per milligram of fetal testis (×104) of Ad spermatogonia (0.42 ± 0.11) was 45% lower (P = 0.03), and that of gonocytes (0.58 ± 0.06) and UI (0.45 ± 0.12) was twofold greater (P < 0.01 and P = 0.06, respectively), than in untreated baboons. Moreover, in the seminiferous cords of estrogen-deprived baboons, the basement membrane appeared fragmented, the germ cells and Sertoli cells appeared disorganized, and vacuoles were present. We conclude that endogenous estrogen promotes fetal testis development and that the changes in the germ cell population in the estrogen-deprived baboon fetus may impair spermatogenesis and fertility in adulthood.
PMCID: PMC2767192  PMID: 19403930
developmental biology; estradiol; Sertoli cells; spermatogenesis; testis
23.  Effects of ETV5 (Ets Variant Gene 5) on Testis and Body Growth, Time Course of Spermatogonial Stem Cell Loss, and Fertility in Mice1 
Biology of reproduction  2007;78(3):483-489.
The transcription factor ets variant gene 5 (ETV5; also known as ERM) is essential for self-renewal of spermatogonial stem cells (SSCs). Mice with targeted disruption of Etv5 (Etv5−/−) undergo the first wave of spermatogenesis, but all SSCs are lost during this time, causing a Sertoli cell-only phenotype. This study examined body and testis growth and the time course of SSC loss in Etv5−/− mice to understand how loss of ETV5 impacts testicular and somatic development. Body weights were reduced in postnatal Etv5−/− males, indicating a role of ETV5 in growth. Testis weights and histology in Etv5−/− and wild-type (WT) males were similar at Postnatal Day 4, but testis weights were reduced at d8 and subsequently, indicating that ETV5 impacts postnatal testis growth. SSC density (SSCs per μm2 of seminiferous tubule), estimated using an antibody against GFRA1, was similar in 4d WT and Etv5−/− mice. By 8 and 12d, GFRA1-positive cell density in Etv5−/− mice was decreased 17% and 32%, respectively, vs. WT. By 28d, GFRA1-positive cell density in Etv5−/− was reduced 95%, and GFRA1-positive cells were absent in 36d Etv5−/− males. In contrast to WT, 35- to 56-day-old Etv5−/− mice were infertile as assessed by natural breeding, artificial insemination, and in vitro fertilization, although motile sperm were present in epididymides of Etv5−/− mice during this time. In summary, initial testis development is normal in Etv5−/− mice despite decreased body weight, but SSC loss begins between 4 and 8d of age, indicating that ETV5 has effects beginning in the early neonatal period. Etv5−/− mice are infertile even when sperm is produced, indicating that ETV5 loss has other effects besides lack of SSC self-renewal that impair fertility.
PMCID: PMC2911230  PMID: 18032421
germ cells; sertoli cells; spermatogenesis; stem cell niche
24.  Gfra1 Silencing in Mouse Spermatogonial Stem Cells Results in Their Differentiation Via the Inactivation of RET Tyrosine Kinase1 
Biology of reproduction  2007;77(4):723-733.
Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate into sperm. The role of growth factor receptors in regulating self-renewal and differentiation of spermatogonial stem cells remains largely unclear. This study was designed to examine Gfra1 receptor expression in immature and adult mouse testes and determine the effects of Gfra1 knockdown on the proliferation and differentiation of type A spermatogonia. We demonstrated that GFRA1 was expressed in a subpopulation of spermatogonia in immature and adult mice. Neither Gfra1 mRNA nor GFRA1 protein was detected in pachytene spermatocytes and round spermatids. GFRA1 and POU5F1 (also known as OCT4), a marker for spermatogonial stem cells, were co-expressed in a subpopulation of type A spermatogonia from 6-day-old mice. In addition, the spermatogonia expressing GFRA1 exhibited a potential for proliferation and the ability to form colonies in culture, which is a characteristic of stem cells. RNA interference assays showed that Gfra1 small interfering RNAs (siRNAs) knocked down the expression of Gfra1 mRNA and GFRA1 protein in type A spermatogonia. Notably, the reduction of Gfra1 expression by Gfra1 siRNAs induced a phenotypic differentiation, as evidenced by the elevated expression of KIT, as well as the decreased expression of POU5F1 and proliferating cell nuclear antigen (PCNA). Furthermore, Gfra1 silencing resulted in a decrease in RET phosphorylation. Taken together, these data indicate that Gfra1 is expressed dominantly in mouse spermatogonial stem cells and that Gfra1 knockdown leads to their differentiation via the inactivation of RET tyrosine kinase, suggesting an essential role for Gfra1 in spermatogonial stem cell regulation.
PMCID: PMC2911237  PMID: 17625109
differentiation; Gfra1; Gfra1 knockdown; mouse; spermatogonial stem cells; testis
25.  Comparative Study of the Dynamics of Follicular Waves in Mares and Women1 
Biology of reproduction  2004;71(4):1195-1201.
Deviation in growth rates of the follicles of the ovulatory wave begins at the end of a common growth phase and is characterized by continued growth of the developing dominant follicle (F1) and regression of the largest subordinate follicle (F2). Follicle diameters during an interovulatory interval were compared between 30 mares and 30 women, using similar methods for collecting and analyzing data. Follicles were tracked and measured daily by ultrasonography. Diameter at follicle emergence (mares, 13 mm; women, 6 mm) and the required minimal attained diameter for assessment of follicles (mares, 17 mm; women, 8 mm) were chosen to simulate the reported ratio between the two species in mean diameter of F1 at the beginning of deviation (mares, 22.5 mm; women, 10.5 mm). F1 emerged before F2 (P < 0.02) in each species, and the interval between emergence of the two follicles was similar (not significantly different) between species. Growth rate for F1 and F2 during the common growth phase was similar within species, and the percentage of diameter increase was similar between species. Proportionality between species in diameter of F1 at deviation (2.2 times larger for mares than for women) and at maximum preovulatory diameter (2.1 times larger) indicated that relative growth of F1 after deviation was similar between species. A predeviation follicle was identified in 33% of mares and 40% of women and was characterized by growth to a diameter similar to F1 at deviation but with regression beginning an average of 1 day before the beginning of deviation. The incidence of a major anovulatory wave preceding the ovulatory wave was not different between species (combined, 25%). Results indicated that mares and women have comparable follicle interrelationships during the ovulatory wave, including 1) emergence of F1 before F2, 2) similar length of intervals between sequential emergence of follicles within a wave, 3) similar percentage growth of follicles during the common growth phase, and 4) similar relative diameter of F1 from the beginning of deviation to ovulation. Similar follicle dynamics between mares and women indicate the mare may be a useful experimental model for study of folliculogenesis in women, with the advantage of larger follicle size.
PMCID: PMC2891974  PMID: 15189824 CAMSID: cams593
follicle; follicular development; follicular waves; mares; ovary; ovulation; ovulatory cycle; women

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