Disruption of Ube2b in the mouse has revealed that the regular and symmetric organization of the fibrous sheath of the sperm flagella is dependent on expression of the ubiquitin-conjugating enzyme UBE2B. These data could cast light on how a component of the ubiquitin-proteasome pathway participates in the assembly of flagellar periaxonemal structures. Data in the literature support the notion of involvement of ubiquitin-proteasome pathways in the assembly of cytoskeletal components in somatic cells. This review attempts to integrate recent knowledge regarding flagellar components that could be related to proteasome components and, therefore, could be targets of UBE2B in the spermatid. An attempt is made to characterize the human flagellar anomalies of infertile patients, which are the closest to those of Ube2b-deficient mice. These new insights regarding the assembly of mammalian sperm flagella provide a basis for studying the ontogenesis of flagellar accessory structures and suggest leads for medical and genetic investigations.

Carbohydrates attached to the protein core of glycoprotein hormones influence a number of intracellular and extracellular processes. As with other members of the glycoprotein hormone family, FSH is produced and released as an array of isoforms that differ from each other in the structure of their oligosaccharide attachments. In this review, we discuss how carbohydrate heterogeneity can impact on FSH action in different in vitro and in vivo systems. We present evidence for diverse effects of distinct charge isoforms at the target cell level, including differential and unique effects on various end responses, and discuss how the use of multiple cell-type assays has allowed identification of some specific effects of FSH isoforms on different cell populations and follicle compartments as well as oocyte maturation. Finally, we discuss recent information on the ability of naturally occurring and laboratory manufactured FSH isoforms to evoke particular effects on granulosa cell function and ovarian follicular maturation in vivo. Such studies have provided evidence that the type(s) of FSH signal delivered may in fact regulate distinct biological outcomes irrespective or in addition to outcomes dictated solely by clearance rate differences.

Limits to estrogen production by early and late preovulatory porcine follicles were assessed by comparing enzymatic capacities for androgen (17,20-lyase) and estrogen (aromatase) synthesis in theca interna and granulosa, support of enzyme activities by the redox partner proteins NADPH-cytochrome P450 oxidoreductase (reductase) and cytochrome b₅, and tissue-specific expression and regulation of these proteins. Parameters included follicular fluid (FF) estradiol and progesterone levels, theca and granulosa aromatase and reductase activities, and theca 17,20-lyase activity. Expression of proteins responsible for these activities, aromatase (P450arom) and 17α-hydroxylase/17,20-lyase (P450c17) cytochromes P450, reductase, and for the first time in ovarian tissues cytochrome b₅, were examined by Western immunoblot and immunocytochemistry. Theca and granulosa aromatase activities were as much as 100-fold lower than theca 17,20-lyase activity, but aromatase was correlated with only the log of FF estradiol. Granulosa reductase activity was twice that of the theca, and cytochrome b₅ expression was clearly identified in both the theca and granulosa layers, as was P450arom, but was not highly correlated with either 17,20-lyase or aromatase activities. Reductase expression did not change with stage of follicular development, but cytochrome b₅, P450c17, and P450arom were markedly lower in post-LH tissues. These data indicate that aromatase and not 17,20-lyase must limit porcine follicular estradiol synthesis, but this limitation is not reflected acutely in FF steroid concentrations. Neither reductase nor cytochrome b₅ appear to regulate P450 activities, but the expression of cytochrome b₅ in granulosa and theca suggests possible alternative roles for this protein in follicular development or function.

Our hypothesis was that luteal function, as determined by plasma progesterone concentrations, and corpus luteum (CL) size is enhanced in cattle administered an agonist of GnRH when the CL is developing as compared with administration of an agonist when the CL is fully functional. Cattle were chronically administered a GnRH agonist, azagly-nafarelin, from Day 3 to Day 21 (D3) or Day 12 to Day 21 (D12) or served as untreated control females (Day 0 = behavioral estrus). Blood samples were serially collected on Days 7 and 14 to evaluate LH secretory patterns and twice daily to measure plasma progesterone. Ultrasonographic examinations were conducted daily to record the area of the CL. CL size and plasma progesterone concentrations were both enhanced in the D3 group as compared with the control group. Progesterone was increased in the D12 group on Days 16 and 17 as compared with the control females. Treatment with GnRH agonist increased basal and mean LH concentrations in both D3 and D12 groups as compared with the controls. We rejected our hypothesis because chronic administration of a GnRH agonist increased plasma progesterone when administered both when the CL was developing and when it was fully functional. The enhanced luteal function was likely due to increased basal LH.

In mice and women, terminal differentiation of uterine natural killer (uNK) cells commences during endometrial decidualization. Both proliferation and interferon (IFN)-γ are induced. Uterine NK cell precursors appear to home from secondary lymphoid organs to decidualizing uteri and localize mesometrially to the central decidua basalis, the site of maternal arterial modification at Gestation Days (gd) 9.5–10. In mice, genetic absence of uNK cells results in absence of pregnancy-induced spiral artery modification. Administration of IFN-γ to uNK-negative pregnant females induces arterial modifications without fetal loss. In this study, we investigated the roles of cytokines, known in other tissues to differentiate and activate NK cells, in induction of IFN-γ production in normal mouse implantation sites. Fecundity evaluation, implantation site morphometry, and IFN-γ quantification in interleukin (IL)-12p40^0/0, IL-18^0/0, dual IL-12p40^0/0/IL-18^0/0 and congenic strains revealed the importance of both IL-12 and IL-18 in the induction of spiral artery modification and IFN-γ synthesis. Immediately after implantation, IL-18 was localized transiently to decidual cells, but by gd8, IL-18 was produced solely by uNK cells, suggesting that early uNK cells are activated by stroma and lymphocyte-derived signals maintain later uNK cell activation. Mesometrial tissue of C57Bl/6J mice was examined by reverse transcription polymerase chain reaction assay in virgin, early postimplantation, and midgestation females for expression of the heterodimeric cytokines IL-23 (composed of IL-12p40 and a novel α chain), IL-27 (composed of two IL-12-related chains) and IL-27R. No expression was detected in virgin uteri. The four genes were induced by gd6, and uNK cells isolated from midgestation transcribed IL-23α and IL-27R. This study advances the understanding of uNK cell activation during normal pregnancy.

Spermatogonial transplantation provides access to the mammalian germline and has been used in experimental animal models to study stem cell/niche biology and germline development, to restore fertility, and to produce transgenic models. The potential to manipulate and/or transplant the germline has numerous practical applications that transcend species boundaries. To make the transplantation technology more broadly accessible, it is necessary to develop practical recipient preparation protocols. In the current study, mouse recipients for spermatogonial transplantation were prepared by treating pregnant females with the chemotherapeutic agent busulfan at different times during gestation. Donor germ cells were introduced into the testes of male progeny between 5 and 12 days postpartum. Analysis of recipient animals revealed that busulfan treatment of pregnant females on 12.5 days postcoitum was the most effective; male progeny transplanted with donor germ cells became fertile and passed the donor genotype to 25% of progeny. This approach was effective because 1) the cytoablative treatment reduced (but did not abolish) endogenous spermatogenesis, creating space for colonization by donor stem cells, 2) residual endogenous germ cells contributed to a healthy testicular environment that supported robust donor and recipient spermatogenesis, and 3) fetal busulfan-treated males could be transplanted as pups, which have been established as better recipients than adults. Laboratory mice provide a valuable experimental model for developing the technology that now can be applied and evaluated in other species.

Differential display in combination with a cDNA cloning approach were used to isolate a novel gene, spergen-2, which has an open reading frame of 1500 nucleotides and encodes a protein of 500 amino acids that contains ankyrin repeat motifs and a putative nuclear localization signal. Expression of spergen-2 is developmentally upregulated in testis. In situ hybridization revealed that spergen-2 mRNA is expressed in spermatocytes and round spermatids (steps 1–6). Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spergen-2 protein is predominantly expressed in nuclei of late spermatocytes (stages IX–XIV) and spermatids (steps 1–11), indicating the restricted expression of spergen-2 during spermatogenesis. In nucleoplasm of spermatogenic cell nuclei, spergen-2 tends to localize in the interchromosome space with relatively low DNA density. These findings indicate a potential role of spergen-2 in spermatogenesis, especially in cell differentiation from late pachytene spermatocytes to spermatids or in early spermatid differentiation.

Cloning by somatic cell nuclear transfer can result in the birth of animals with phenotypic and gene expression abnormalities. We compared adult cloned pigs and adult pigs from naturally bred control females using a series of physiological and genetic parameters, including detailed methylation profiles of selected genomic regions. Phenotypic and genetic analyses indicated that there are two classes of traits, one in which the cloned pigs have less variation than controls and another characterized by variation that is equally high in cloned and control pigs. Although cloning creates animals within the normal phenotypic range, it increases the variability associated with some traits. This finding is contrary to the expectation that cloning can be used to reduce the size of groups involved in animal experimentation and to reproduce an animal, including a pet, with a homogenous set of desired traits.

The production of genetically engineered pigs as xenotransplant donors aims to solve the severe shortage of organs for transplantation in humans. The first barrier to successful xenotransplantation is hyperacute rejection (HAR). HAR is a rapid and massive humoral immune response directed against the pig carbohydrate Galα1,3-Gal epitope, which is synthesized by α1,3-galactosyltransferase (α1,3-GT). The Galα1,3-Gal antigen also contributes to subsequent acute vascular rejection events. Genetic modifications of donor pigs transgenic for human complement regulatory proteins or different glycosyltransferases to downregulate Galα1,3-Gal expression have been shown to significantly delay xenograft rejection. However, the complete removal of the Galα1,3-Gal antigen is the most attractive option. In this study, the 5′ end of the α1,3-GT gene was efficiently targeted with a nonisogenic DNA construct containing predominantly intron sequences and a Kozak translation initiation site to initiate translation of the neomycin resistance reporter gene. We developed two novel polymerase chain reaction screening methods to detect and confirm the targeted G418-resistant clones. This is the first study to use Southern blot analysis to demonstrate the disruption of the α1,3-GT gene in somatic HT-transgenic pig cells before they were used for nuclear transfer. Transgenic male pigs were produced that possess an α1,3-GT knockout allele and express a randomly inserted human α1,2-fucosylosyltransferase (HT) transgene. The generation of homozygous α1,3-GT knockout pigs with the HT-transgenic background is underway and will be unique. This approach intends to combine the α1,3-GT knockout genotype with a ubiquitously expressed fucosyltransferase transgene producing the universally tolerated H antigen. This approach may prove to be more effective than the null phenotype alone in overcoming HAR and delayed xenograft rejection.

Sperm adhesion molecule 1 (Spam1) is a widely conserved sperm surface protein with multiple roles in mammalian fertilization. Although the gene for this protein has been thought to be testis specific based on Northern blot analysis, there is evidence for nontesticular expression when transcripts are analyzed by more sensitive techniques. In the present investigation, results of a reverse transcription polymerase chain reaction assay, an RNase-protection assay (RPA), and an in situ transcript hybridization assay revealed that the murine Spam1 gene is transcribed in the female genital tract. RPA revealed that Spam1 transcripts are synthesized in a region-dependent manner, with the oviduct having lower transcript levels than the uterus and vagina. The transcripts levels were 3- to 10-fold lower in the female genital tract than in the testis. In situ transcript hybridization assay revealed RNA in the luminal epithelium in all three regions of the genital tract and in the uterine myometrium and the oviductal mesothelium. Western blot analysis and immunohistochemistry demonstrated that the protein concentration is 1.5- to 3-fold lower in female tissues than in sperm, and localization is similar to that of the transcripts. The protein has hyaluronidase activity at neutral pH, which is unique for sperm hyaluronidase, but not at acidic pH. In the uterus, Spam1 expression fluctuated during the estrous cycle. Its localization suggests that in addition to functioning as a secretory protein, it may be involved in hyaluronic acid metabolism or turnover in the female genital tract. Our results provide further evidence that Spam1 is a multifunctional protein and that it is less restricted in its expression than previously reported.

Black porgy, Acanthopagrus schlegeli Bleeker, is a marine protandrous hermaphrodite fish. All are functional males at 1–2 yr of age and then become either males or females at 3 yr of age. To study the process of sex change in this species, mRNA transcripts of two estrogen receptors (ERα and ERβ) and an androgen receptor (AR) were monitored. An AR cDNA was cloned and characterized. ERα, ERβ, and AR were differentially transcribed in bisexual testicular and ovarian tissue according to reverse transcription polymerase chain reaction (RT-PCR) and Southern analysis. A real-time quantification PCR analysis was further developed for the measurement of AR, ERα, and ERβ transcripts. ERα and AR transcripts were significantly more plentiful in bisexual testis than in bisexual ovary in 1 - and 2 -yr-old fish. ERα, ERβ, and AR transcripts decreased in the functional testis of 3-yr-old fish. Similar levels of ERβ and AR were detected in the ovary of sex-changed females and in functional testis of 3-yr-old males. Significantly decreased AR transcripts were found in testicular tissue of bisexual and functional male and female gonads in 3-yr-old fish as compared with 1- and 2-yr-old fish. In contrast, increased ERα transcripts were detected in the bisexual ovary and sex-changed ovary of 3-yr-old fish as compared with the bisexual ovary of 1- and 2-yr-old fish. The data suggest a differential sensitivity in the bisexual testicular and ovarian tissue of black porgy.

Alkylphenolic compounds are a widespread family of xenoestrogens. High concentrations of these substances are present in sewage sludge that is spread on arable land and pasture as fertilizer. Because of their known endocrine system-disrupting activity, alkylphenols represent a potential risk for the reproductive health of farm animals. In this study, the impact of p-tert-octylphenol (OP) on the developmental competence of bovine oocytes was evaluated. Endocrine activity of OP was investigated for its effect on estrogen receptors α and β (ERα and ERβ) and progesterone receptor (PR) mRNA levels. Cumulus-oocyte complexes (COCs) were exposed during in vitro maturation to serial concentrations of OP (1–0.0001 μg/ml) and were compared with vehicle-treated controls and a group of COCs treated with 17β-estradiol (E₂). A dose-related decrease in the percentage of oocytes that completed maturation after 24 h and in oocyte fertilization competence was observed at doses of OP as low as 0.01 μg/ml. Groups treated with ≥0.001 μg/ml OP showed impaired embryo development. No adverse effects of E₂ were observed. In the E₂-treated COCs, ERα mRNA was decreased but PR mRNA was upregulated compared with controls. Treatment with 0.001 and 0.0001 μg/ml OP induced a decrease in ERα mRNA, but ERβ and PR mRNA were not affected. Treatment with 0.01 μg/ml OP did not produce changes in the expression of any of the mRNAs studied. OP impairs meiotic progression and developmental competence of bovine oocytes without demonstrating clear estrogen-mimic activity.

Turkey sperm lose viability within 8–18 h when stored as liquid semen using current methods and extenders. In contrast, turkey hens maintain viable, fertile sperm in their sperm storage tubules (SST) for 45 or more days following a single insemination. Our long-term objectives are to identify and characterize differentially expressed genes that may underlie this prolonged sperm storage and then use this information to develop improved methods for storing liquid turkey semen. We employed serial analysis of gene expression (SAGE) to compare gene expression patterns in turkey SST recovered from hens after artificial insemination (AI) with extended semen (sperm AI) or extender alone (control AI). We constructed two separate SAGE libraries with SST RNA obtained from sperm and control AI hens. We used these libraries to generate 95 325 ten-base pair SAGE tags. These 95 325 tags represented 27 430 unique genes. The sperm and control AI libraries contained 47 663 and 47 662 tags representing 18 030 and 19 101 putative unique transcripts, respectively. Approximately 1% of these putative unique genes were differentially expressed (P < 0.05) between treatments. Tentative annotations were ascribed to the SAGE tag nucleotide sequences by comparing them against publicly available SAGE tag and cDNA sequence databases. Based on its SAGE tag nucleotide sequence, we cloned a partial turkey avidin cDNA and confirmed its up-regulation in the sperm AI SST. The bioinformatics and experimental procedures employed to clone turkey avidin and confirm its differential expression represent a useful paradigm for analyzing SAGE tag data from relatively uncharacterized model systems.

We isolated cDNA clones for the novel actin-like proteins T-ACTIN 1 and T-ACTIN 2, which are expressed specifically in the mouse testis. These clones were from a subtracted cDNA library that was enriched for haploid germ cell-specific cDNAs. The mRNA sizes and deduced molecular masses of t-actin 1/mACTl7b and t-actin 2/mACTl7a were 2.2 kilobases (kb) and 1.8 kb, and M_r 43.1 × 10³ and M_r 47.2 × 10³, respectively. The two deduced amino acid sequences had 60% homology, and they had approximately 40% homology with other actins. The T-ACTINs contained some of the conserved regions seen in other actins. Although the cellular locations of these two proteins are quite different (T-ACTIN-1 was found in the cytoplasm and T-ACTIN-2 was located in the nucleus), the expression of their proteins and mRNAs is controlled during development and limited during spermiogenesis. In contrast, only T-ACTIN-2 was present in sperm heads and tails. These results suggest that T-ACTINs play important roles in sperm function and in the specific morphogenesis of spermatozoa during spermiogenesis.

Mice of the XO genotype with a paternally derived X chromosome (X^pO) have placental hyperplasia in late pregnancy, although in early pregnancy the ectoplacental cone, a placental precursor, is smaller in X^pO mice than in their XX sibs. This early size deficiency of the ectoplacental cone is apparently a consequence of X^p imprinting, because X^mO embryos (with a maternally derived X chromosome) are unaffected. In the present study we sought to establish whether X^pO placental hyperplasia in late pregnancy is also a consequence of X^p imprinting. Placental weight data were first collected from litters that included X^pO or X^mO fetuses and XX controls. Comparison of XO placentae with XX placentae showed that X^pO and X^mO placentae are hyperplastic. This finding suggested that the hyperplasia might be an X dosage effect, and this hypothesis was supported by the finding that XY male fetuses from the same crosses also had larger placentae than their XX sibs. Further analysis of a range of sex-chromosome variant genotypes, including X^mYSry-negative females and XXSry transgenic males, showed that mouse fetuses with one X chromosome consistently had larger placentae than littermates with two X chromosomes, independent of their gonadal/androgen status.

The family of type 2 cystatin proteins is a class of cysteine proteinase inhibitors that function as potent inhibitors of papain-like cysteine proteinases. Recent studies have suggested that cystatins in the male reproductive tract subgroup may perform functions distinct from those of typical cystatins. The objective of the present study was to identify and characterize the expression of new gene members of the cystatin family 2 in mouse male reproductive tissues. Two new members of cystatin family 2, named mouse Cystatin E1 and mouse Cystatin E2 (mCST E1 and mCST E2, respectively), were identified in mice by searching the National Center for Biotechnology Information database for proteins containing homology to known type 2 cystatins. Human CST E1 has recently been reported independently under the name CST 11. The deduced amino acid sequences of these genes have significant homology with the family 2 cystatins, including four conserved cysteine residues at the C-terminus. Similar to other male reproductive subgroup cystatins, the inhibitory motifs are not well conserved in these genes. Northern blot analyses showed that both genes were highly expressed only in the epididymis. In situ hybridization demonstrated that both genes were restricted in their expression to the epithelial cells of the caput and that the highest expression was localized to the initial segment of caput epididymis. Northern blot analyses and in situ hybridization showed that both mCST E1 and E2 mRNA decreased after castration, and treatment with testosterone propionate (T) did not maintain expression of these genes. In fact, T treatment further repressed the expression of these genes in the epididymis following castration. Efferent ductule ligation resulted in a dramatic decrease of epididymal expression of mCST E1 and E2. The expression of mCST E1 mRNA was up-regulated by 17β-estradiol (E) administration for 7 days postcastration, whereas no recovery of mCST E1 mRNA level was detected after 14 days of E treatment. Combined E and T (E T) treatment for 1 and 2 wk reduced the mCST E1 transcripts. The expression of mCST E2 mRNA was maintained by E administration for both 7 and 14 days after castration, whereas treatment of both T and E repressed the expression of mCST E2. Although both mCST E1 and E2 share significant homology with family 2 cystatins, including similar distribution in tissues and localization in epididymis, these genes may have different functions, because their regulation involves different hormones and, probably, other testicular factors.

Apoptosis occurs during preimplantation development in both in vivo- and in vitro-produced embryos, and it may contribute to embryonic loss. The present study investigated the development of porcine nuclear transfer (NT) embryos reconstructed by using fetal fibroblasts as compared to embryos produced by in vitro fertilization (IVF). The onset and the frequency of apoptosis in NT and IVF embryos were examined via morphological and nuclear changes and TUNEL assay. The NT blastocysts had a similar number of nuclei as compared to IVF blastocysts and appeared to be morphologically similar. Relative to IVF embryos, the NT embryos had a lower cleavage rate (42.7% vs. 71.0%) and a lower developmental rate (11.1% vs. 28.6%) to the blastocyst stage. The earliest positive TUNEL signals were detected in the NT embryos on Day 5 of culture. The percentage of cells undergoing apoptosis in the NT embryos was higher than that of the IVF embryos and increased with time in vitro. Some of the abnormal morphological changes observed during early development related to apoptosis. Cytoplasmic fragmentation, developmental arrest, and nuclear condensation were typical characteristics of embryos undergoing apoptosis. Some mechanisms of the apoptotic pathway were triggered by changes in the NT embryos. The developmental rates of NT embryos might be improved by identifying specific apoptotic pathways and then intervening in these pathways to improve development.

Liver receptor homologue 1 (LRH-1) is a member of the nuclear receptor superfamily originally found in liver cells. LRH-1 participates in regulation of cholesterol metabolism and bile acid synthesis. Recent studies have shown that LRH-1 is even more highly expressed in the ovary, and LRH-1 has been implicated as a key transcriptional regulator of cytochrome P450 aromatase (P450_arom) in vitro. In the present study, we investigated the spatiotemporal expression patterns of LRH-1 using in situ hybridization and immunohistochemistry in ovaries from rats with a 4-day estrous cycle, from pregnant rats, from immature rats treated with eCG to stimulate follicular development, and from eCG-treated rats that were subsequently given hCG to stimulate ovulation and luteinization. To establish a potential connection between the expression of LRH-1 and that of the steroidogenic genes in vivo, we directly compared the localization patterns of LRH-1 and P450_arom transcripts in consecutive ovarian sections from these animals. LRH-1 mRNA and protein were primarily localized to granulosa cells and luteinized follicles or newly formed corpora lutea (CLs) of immature and adult rats, and the levels of expression increased during eCG-hCG-induced follicular development and ovulation. In the functional CLs of pregnant rats, a biphasic change in LRH-1 mRNA content occurred throughout the gestation process, whereas LRH-1 protein was persistently detected during the entire pregnancy. In the consecutive ovarian sections, expression of LRH-1 was approximately colocalized with that of P450_arom in both tertiary and Graafian follicles and the functional CLs of pregnant rats. LRH-1 mRNA and protein expression preceded those of P450_arom during early follicular development. Stage-specific expression of LRH-1 in rat granulosa and luteal cells suggests a role for LRH-1 in the regulation of ovarian function. The overlapping but distinct expression patterns of LRH-1 and P450_arom circumstantially support the recent finding that LRH-1 serves as a critical upstream regulator of P450_arom gene expression in ovarian cells, but LRH-1 also may be a multifunctional steroidogenic factor in ovarian physiology.

Colony-stimulating factor 1 (CSF-1) is a hematopoetic cytokine that also plays an important role in placental physiology. We report here the molecular cloning of two alternative splice variants of the bovine gene coding for a putative secreted and a membrane-bound form of the cytokine and the dynamic regulation of expression in the reproductive tract of cattle during the estrous cycle and pregnancy. Bovine CSF-1 was expressed mainly as the 3- and 4-kilobase (kb) transcripts, but 1.4- and 0.8-kb mRNAs were also detected in Day 50–70 pregnant uterine tissue. During the estrous cycle, both the 4- and 3-kb mRNAs were present, but the 3-kb putative membrane-bound form was more abundant than the 4-kb secreted form during diestrus. This pattern of expression was reversed in pregnancy, so that the exponential increase in CSF-1 expression seen during pregnancy was due predominantly to increased abundance of the 4-kb transcript. The change in the 4-kb:3-kb ratio was detected between Day 14 and Day 17, approximately the time of maternal recognition of pregnancy. Thus, CSF-1 was identified as one gene whose expression in the uterus might be altered early in response to the presence of the conceptus. CSF-1 was also expressed in the extraembryonic membranes of the conceptus and in the trophoblastic cells of the fetal cotyledons after the formation of the placentomes. The high level of CSF-1 expression during bovine pregnancy in uteroplacental tissues is consistent with its proposed role in placental physiology.

Ejaculated mammalian sperm must undergo a final maturation (capacitation) before they can acrosome-react and fertilize eggs. Loss of the sperm sterols, cholesterol and desmosterol, is an obligatory step in the capacitation of human sperm. Because sterols can increase the order of membrane phospholipids, it has been suggested that the importance of sterol loss is that it decreases membrane lipid order. The present study tested the hypotheses that sterol loss decreases sperm membrane lipid order during capacitation and that lipid disorder is a sufficient stimulus for capacitation. Steady-state fluorescence anisotropy of the membrane probe, 1,6-diphenyl-1,3,5-hexatriene, decreased during capacitation, indicating a decrease in lipid order. The decrease was dependent on the loss of sperm sterols, suggesting that it reflected diminished sterol-mediated phospholipid ordering. However, the lipid-fluidizing agents, benzyl alcohol and 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) octanoate, did not cause sperm capacitation or overcome inhibition by cholesterol. In summary, loss of sperm sterols caused a significant decline in lipid order during capacitation; however, decreased bulk lipid order was not sufficient to trigger the subsequent events that complete capacitation.

Infertile men undergoing intracytoplasmic sperm injection have an increased frequency of chromosome abnormalities in their sperm. Men with low sperm concentration (oligozoospermia) have an increased risk of sperm chromosome abnormalities. This study was initiated to determine whether men with severe oligozoospermia (<10⁶ sperm/ml) have a higher frequency of chromosome abnormalities in their sperm compared with men with moderate (1–9 × 10⁶ sperm/ml) or mild (10–19 × 10⁶ sperm/ml) oligozoospermia. Multicolor fluorescence in situ hybridization analysis was performed using DNA probes specific for chromosomes 13, 21, X, and Y (with chromosome 1 as an autosomal control for the sex chromosomes). Aneuploidy and disomy frequencies were assessed from a total of 603 011 sperm from 30 men: 10 in each of the categories. The mean frequencies of disomy for the patients with mild, moderate, and severe oligozoospermia were 0.17%, 0.24%, and 0.30%, respectively, for chromosome 13 and 0.22%, 0.44%, and 0.58%, respectively, for chromosome 21. For the sex chromosomes, the mean frequencies of disomy for mild, moderate, and severe oligozoospermia were 0.25%, 1.04%, and 0.68%, respectively, for XY, 0.047%, 0.08%, and 0.10%, respectively, for XX, and 0.04%, 0.06%, and 0.09%, respectively, for YY. The frequencies for diploidy also increased from 0.4% for mild to 1.20% for moderate to 1.24% for severe oligozoospermia. There was a significant inverse correlation between the frequency of sperm chromosome abnormalities and the sperm concentration for XY, XX, and YY disomy and diploidy. These results demonstrate that men with severe oligozoospermia have an elevated risk for chromosome abnormalities in their sperm, particularly sex chromosome abnormalities.

In pregnant mares during late gestation, little, if any, progesterone (P₄) is found in the maternal circulation. Hence, quiescence of the equine uterus is believed to be maintained by metabolites of pregnenolone and P₄ known as progestagens, which are produced by the uteroplacental tissues. However, little is known about the ontogeny, distribution, or actual rates of uteroplacental progestagen production in pregnant mares and their fetuses during the second half of pregnancy. Therefore, the present study measured the rates of uteroplacental uptake and output of eight specific progestagens in chronically catheterized, pregnant pony mares from 180 days to term. No significant uteroplacental uptake of any of the eight individual progestagens was observed from the uterine circulation. In contrast, significant uteroplacental uptake was observed for five of the eight individual progestagens from the umbilical circulation, and the uptakes increased toward term. The major uteroplacental progestagen outputs were 5α-pregnane-3,20-dione (5αDHP) and 20α-hydroxy-5α-pregnan-3-one (20α5P). These were released into both the umbilical and uterine circulations at rates that increased toward term. The majority of the total uteroplacental 20α5P output was distributed into the uterine circulation at all gestational ages studied. In contrast, distribution of the total uteroplacental 5αDHP output switched from preferential delivery into the uterine circulation before 220 days of gestation to release predominantly into the umbilical circulation after 260 days. These findings demonstrate that uteroplacental progestagen production changes during the second half of gestation, which may have important implications for the maintenance of pregnancy and the onset of labor in the mare.

Messenger RNA for peroxisome proliferator-activated receptor γ (PPARγ) has been found in granulosa cells, and its expression decreases after the LH surge. We determined which developmental stage of ovarian follicle expresses mRNA for PPARγ and evaluated the impact of PPARγ agonists on steroidogenesis. Ovaries were collected from immature eCG/hCG-treated rats at 0 (no eCG), 24, and 48 h post-eCG and 4 and 24 h post-hCG. Ovarian tissue was serially sectioned and processed for in situ hybridization to localize mRNA corresponding to PPARγ, aromatase, and the LH receptor, and P450 side chain cleavage (P450_SCC) and to determine whether apoptotic cells were present. During follicular development, there was no correlation between the expression of mRNAs for PPARγ and aromatase or the presence of apoptotic cells, but a general inverse correlation was observed between the expression of PPARγ mRNA and LH receptor mRNA. At 4 h post-hCG, follicles expressing P450_SCC mRNA had lost expression of PPARγ mRNA. This inverse pattern of expression between PPARγ and P450_SCC mRNAs was also observed 24 h post-hCG, with developing luteal tissue expressing high levels of P450_SCC mRNA but little or no PPARγ mRNA. To determine the impact of PPARγ on steroidogenesis, granulosa cells were collected from ovaries 24 h post-eCG and cultured alone, with FSH alone, or with FSH in combination with the PPARγ agonists ciglitazone or 15-deoxy-Δ^12,14-prostaglandin J₂ (PGJ₂). Treatment of granulosa cells with PGJ₂ stimulated basal progesterone secretion, whereas ciglitazone or PGJ₂ had no significant effect on FSH-stimulated steroid production. These findings suggest that 1) PPARγ may regulate genes involved with follicular differentiation and 2) the decline in PPARγ in response to LH is important for ovulation and/or luteinization.

Myometrial quiescence during pregnancy is maintained by progesterone, which suppresses the expression of labor-associated genes such as connexin 43 (Cx43) and the oxytocin receptor (OTR). Parathyroid hormone-related protein (PTHrP) is a smooth muscle relaxant that inhibits myometrial contractions and therefore may act in synergy with progesterone to maintain myometrial quiescence during late pregnancy. We investigated the possibility that PTHrP, like progesterone, could act to suppress the expression of labor-associated genes. Pregnant rats were treated starting on Day 19 with daily i.p. injections of 100 μg/kg PTHrP (human synthetic fragment 1-34). On Day 22 of gestation, there was a significant reduction in the expression of Cx43 (mRNA and protein) and OTR (mRNA) in the myometrium of PTHrP-treated animals, whereas on Day 23 (labor) the expression of both Cx43 and OTR was unchanged by PTHrP treatment. Treatment of pregnant rats with PTHrP did not affect the time of delivery, concentrations of progesterone in maternal plasma, or levels of c-fos, fra-2, or parathyroid hormone/PTHrP receptor mRNA on any gestational day. Because PTHrP treatment delayed the dramatic increase in the expression of Cx43 and OTR, it may be an important factor in the maintenance of the quiescent state of the myometrium at a time when the concentrations of progesterone in maternal circulation decrease. PTHrP treatment did not prevent the increase in Cx43 and OTR gene expression on Day 23 or the timing of labor, suggesting that the effects of PTHrP signaling are overridden with the onset of labor.

The integrin and extracellular matrix protein (ECM)-mediated adhesion and invasion of the receptive maternal uterine endometrium by trophoblasts is a critical event in the complex physiological process of pregnancy. Although the process has been largely characterized in mice, the relevant mechanism in primates remains unclear. We investigated the expression patterns and dynamic alterations of integrin subunits (α₁, α₅, α₆, β₁, and β₄) and their ECM ligands, such as laminin (LN), type IV collagen (Col IV), and fibronectin (FN), at the maternal-fetal interface during Gestational Days 15, 25, 50, and 100 and at full term in 20 pregnant rhesus monkeys. Immunohistochemistry and in situ hybridization revealed that a relatively high expression of integrins occurred in trophoblast cells at Gestational Day 15, with the peak level occurring at Day 25. The expression level decreased from Day 50 to term. Along the invasive pathway, expression levels of integrin α₁, α₅, and β₁ subunits were gradually elevated from the proximal to distal column, reaching peak level in the trophoblast shell, but were reduced in those invasive extravillous cytotrophoblast (EVCT) cells in contact with the decidua. Integrin α₁, α₅, β₁, and β₄ subunits were also highly expressed in decidual stromal cells and moderately expressed in the maternal epithelium and endothelium. Immunoreactive FN, LN, and Col IV were distributed in EVCT and decidual stromal cells and part of the uterine epithelial and endothelial cells. These data suggest that the correlated expression of integrins and their ECM ligands at the maternal-fetal interface might be involved in regulation of cell proliferation and differentiation and the counterbalanced invasion-accelerating and invasion-restraining processes in trophoblast cells during the early stage of pregnancy.

Ejaculated sperm are unable to fertilize an egg until they undergo capacitation. Capacitation results in the acquisition of hyperactivated motility, changes in the properties of the plasma membrane, including changes in proteins and glycoproteins, and acquisition of the ability to undergo the acrosome reaction. In all mammalian species examined, capacitation requires removal of cholesterol from the plasma membrane and the presence of extracellular Ca² and HCO₃⁻. We designed experiments to elucidate the conditions required for in vitro capacitation of rat spermatozoa and the effects of Crisp-1, an epididymal secretory protein, on capacitation. Protein tyrosine phosphorylation, a hallmark of capacitation in sperm of other species, occurs during 5 h of in vitro incubation, and this phosphorylation is dependent upon HCO₃⁻, Ca² , and the removal of cholesterol from the membrane. Crisp-1, which is added to the sperm surface in the epididymis in vivo, is lost during capacitation, and addition of exogenous Crisp-1 to the incubation medium inhibits tyrosine phosphorylation in a dose-dependent manner, thus inhibiting capacitation and ultimately the acrosome reaction. Inhibition of capacitation by Crisp-1 occurs upstream of the production of cAMP by the sperm.

The succession in time and space of specific germ cell associations, denoted as spermatogenic stages, is a typical feature of mammalian spermatogenesis. The arrangement of these stages is either single stage (one spermatogenic stage per tubular cross-section) or multistage (more than one spermatogenic stage per tubular cross-section). It has been proposed that the single-stage versus multistage arrangement is related to spermatogenic efficiency and that the multistage arrangement is typical for hominids. In the present work, the arrangement of spermatogenic stages and the spermatogenic efficiency of 17 primate species, comprising Strepsirrhini (Prosimians: Lemuriformes, Lorisiformes), Platyrrhini (New World primates), Catarrhini (Old World primates), and Hominoidea (great apes and humans), were analyzed comparatively by quantitative histological and flow cytometric means. We found a predominant single-stage tubular organization in the Strepsirrhini, indicating that the single-stage form represents the ancestral state. The highest degree of multistage complexity was found in Hominoidea (except orangutan) and in Platyrrhini, but not in Catarrhini. Hence, no direct relationship between single-stage/multistage tubular topography and phylogeny could be established across primates. In fact, the tubule arrangement seen in Platyrrhini and Catarrhini primates is the reverse of what might be expected from phylogeny. Interestingly, spermatogenic efficiency was similar in all species. We found no correlation between single-stage/multistage arrangement and spermatogenic efficiency or mating system. We speculate that the presence of a single-stage/multistage organization might simply reflect germ cell clonal size. Our findings further indicate that sperm competition in primates is not reflected at the level of testicular function.

Estrogens are key regulators of sexual differentiation and development in vertebrates. The P450 aromatase (P450arom) is the steroidogenic enzyme responsible for the synthesis of estrogens from androgens. In the adult rat testis, aromatase transcripts and activity have been observed in somatic cells and germ cells, including pachytene spermatocytes (PS) and round spermatids (RS), but little is known concerning regulation of the aromatase gene expression, especially in germ cells. The quality of germ cell preparations was assessed by the absence of androgen-binding protein and stem cell factor transcripts, two specific markers for Sertoli cells. By employing a competitive quantitative reverse transcriptase-polymerase chain reaction technique, we confirmed that germ cells contained P450arom transcripts and demonstrated that the aromatase gene was up-regulated by cAMP. Conversely, transforming growth factor (TGF) β1 inhibited Cyp19 gene expression in a dose- and a time-dependant manner in both PS and RS. The addition of tumor necrosis factor (TNF) α to purified germ cells induced an increase of the amount of P450arom mRNA in PS, although an inhibitory effect was observed in RS. When PS were treated with dexamethasone (Dex), a similar enhancement of the aromatase transcript level was observed, whereas an inhibitory effect was recorded for RS. Furthermore, in either TGFβ1- or TNFα-treated germ cells, the addition of Dex stimulated the aromatase gene transcription. Experiments using 5′ rapid amplification of cDNA ends suggested that promoter PII is mainly concerned in the regulation of the aromatase gene expression in germ cells of adult male rats; however, the presence of other promoters could not be excluded.

Long-term storage of semen by cryopreservation, with high recovery rates on thawing, is essential for the establishment of genetic resource banks of endangered species. The purpose of the present study was to evaluate various diluents for the cryopreservation of spermatozoa from three species of gazelles (genus Gazella) in a captive breeding program. The diluents compared were Tes (N-tris(hydroxymethyl)methyl-2 aminoethane sulfonic acid)-Tris with 5% egg yolk and 6% glycerol (TEST) and Triladyl, yolk-citrate, Tris-trehalose, and Tris-lactose—all of them with 20% egg yolk and 6% (Triladyl) or 8% glycerol. Semen was obtained by electroejaculation from 12 G. cuvieri, 12 G. dama, and 13 G. dorcas males. Samples with less than 50% motile sperm, positive endosmosis, or acrosome integrity were not used. Diluted samples were loaded into 0.25-ml straws, cooled slowly to 5°C over 1.5 h (−0.16°C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapors for 10 min, and plunged into liquid nitrogen. Subsamples were assessed fresh, after refrigeration-equilibration, after freezing and thawing, and 2 h after thawing. Differences were seen between diluents, with best overall recovery rates after freezing and thawing found with Triladyl, TEST, and Tris-trehalose in G. cuvieri, TEST in G. dama, and Triladyl and TEST in G. dorcas. Differences were observed between species in the ability to withstand freezing and thawing, with best results seen in G. dorcas, intermediate results in G. dama, and worst results in G. cuvieri. These differences were inversely related to the average values of inbreeding of these populations. The underlying mechanism responsible for these differences may be a differential resistance to osmotic shock.

Spermatogenesis is a complex process that originates in a small population of spermatogonial stem cells. Here we report the in vitro culture of spermatogonial stem cells that proliferate for long periods of time. In the presence of glial cell line-derived neurotrophic factor, epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory factor, gonocytes isolated from neonatal mouse testis proliferated over a 5-month period (>10¹⁴-fold) and restored fertility to congenitally infertile recipient mice following transplantation into seminiferous tubules. Long-term spermatogonial stem cell culture will be useful for studying spermatogenesis mechanism and has important implications for developing new technology in transgenesis or medicine.

An understanding of testicular physiology and pathology requires knowledge of the regulation of cell death. Previous observation of suppression of apoptosis by hypoxia suggested a role for ATP in germ cell death. However, the exact effects of ATP production on germ cell death and of apoptosis on the levels of ATP and other adenine nucleotides (ANs) have remained unclear. We investigated the levels of ANs during human testicular apoptosis (analyzed by HPLC) and the role of chemical anoxia in germ cell death (detected by Southern blot analysis of DNA fragmentation, in situ end labeling of DNA, and electron microscopy). Incubation of seminiferous tubule segments under serum-free conditions induced apoptosis and concomitantly decreased the levels of ANs. Chemical anoxia, induced with potassium cyanide (KCN), an inhibitor of mitochondrial respiration, dropped ATP levels further and suppressed apoptosis at 4 h. After 24 h, many of the testicular cells underwent delayed apoptosis despite ATP depletion. Some cells showed signs of necrosis or toxicity. The addition of 2-deoxyglucose, an antimetabolite of glycolysis, did not alter the results obtained with KCN alone, whereas a toxic concentration of hydrogen peroxide switched apoptosis to necrosis. In most of the testicular cells, mitochondrial respiration appears to play a crucial role in controlling primary cell death cascades. In the human testis, there seem to be secondary apoptotic pathways that do not require functional respiration (or ATP).

The trophoblast, the outermost layer of the human placenta, lacks expression of the classical human leukocyte antigen (HLA) class I molecules. This prevents allorecognition by T cells but raises the question of what protects the trophoblast from natural killer (NK) cells. In a previous study, we have shown that choriocarcinoma cell (CC) resistance to NK lysis was mainly independent of HLA class I molecules. In the present study, we postulated that CC may prevent activation of NK cells by failing to stimulate their triggering receptors (TR). To test this hypothesis, we evaluated the lysis of JAR and JEG-3 CC after effective cross-linking and activation of NK cells by means of lectins or antibodies. Our results show that NK-resistant CC were sensitive to lysis by unstimulated peripheral blood lymphocytes in the presence of phytohemagglutin (PHA), to antibody-dependent cell cytotoxicity in presence of anti-Tja antibodies, and to monoclonal antibody redirected killing using anti-TR antibodies anti-CD16 and anti-CD244/2B4. Finally, CC fail to express CD48, the ligand for CD244/2B4. These results indicate that the resistance of CC to lysis results primarily from defective NK cell activation, at least partially due to the lack of expression of ligands, such as CD48, involved in the triggering of NK cells.

To date, it has not been possible to detect corpus luteum (CL) by ultrasonography, immediately following ovulation, in the ewe. Early CL detection is essential to be able to relate luteal outcome to the developmental pattern of the ovulated follicle and to confirm ovulation. Image analysis of the CL may be useful in providing a noninvasive picture of CL differentiation and function. The present study was designed to use high-resolution ultrasonography to monitor and to correlate the echotextural, histological, and functional attributes of the developing ovine CL from Days 1 to 3 after ovulation. Ten ewes underwent twice-daily transrectal ultrasonography and blood sampling from the day of synchronized estrus. Ewes were ovariectomized at 12–24, 36–48, and 60–72 h after ovulation. Ovaries collected were scanned in a water bath before processing for histology. Ultrasonographic images of CL were analyzed for echotexture. Histological sections were analyzed for the percentage area of the CL occupied by blood clot or luteal tissue. Serum samples were analyzed for progesterone concentration. Numerical pixel value, heterogeneity, and percentage of the CL occupied by blood clot declined (P < 0.05) from 12–24 to 60–72 h after ovulation. Luteal area and serum progesterone concentration increased (P < 0.05) from 12–24 to 60–72 h. The results indicated that it was possible to visualize developing CL as early as 12–24 h after ovulation in the ewe. Echotexture of the CL was closely associated with its morphological and functional characteristics; image analysis holds promise for noninvasive monitoring of CL differentiation and growth.

The effect of heparin-binding and non-heparin-binding spermadhesins on the viability, motility, and mitochondrial activity of boar spermatozoa at the high dilution (300 000 sperm/ml) to which sperm are exposed during the process of sex sorting by flow cytometry was investigated. Incubation of spermatozoa with heparin-binding spermadhesins caused a time- and dose-dependent decrease in the percentage of functional spermatozoa. The percentage of viable spermatozoa incubated at 38°C with heparin-binding spermadhesins diluted in PBS (1 mg/ml) dropped from 75% (0.5 h) to 4% (5 h), whereas the percentage of viable spermatozoa incubated in PBS without proteins (control) decreased from 85% (0.5 h) to 19% (5 h). Addition of non-heparin-binding PSP-I/PSP-II spermadhesin to the PBS resulted in a concentration-dependent increment of the percentage of viable cells (65% after 5-h incubation), with maximum effect at 1.5 mg/ml. The heparin-binding spermadhesins totally suppressed sperm motility and mitochondrial activity after 5 h of incubation. The same parameters of sperm incubated in the presence of 1.5 mg/ml of PSP-I/PSP-II were 50% and 58%, respectively, and the percentages of control sperm displaying motility and mitochondrial activity were 21% and 26%, respectively. Moreover, the viability, motility, and mitochondrial activity all decreased on incubation of spermatozoa with mixtures of PSP-I/PSP-II and heparin-binding spermadhesins as the concentration of the latter increased. We conclude that PSP-I/PSP-II and the heparin-binding spermadhesins exert antagonistic effects on the functionality of highly diluted boar spermatozoa. The finding that PSP-I/PSP-II contributes to maintaining sperm with high viability, motility, and mitochondrial activity for at least 5 h at physiological temperature points to its potential use as an additive for sperm preservation, specifically of highly diluted, flow-sorted spermatozoa for sex preselection.

Transient receptor potential (Trp) channels have been implicated in mediating store- and receptor-activated Ca² influx. Different properties of this influx in various cell types may stem from the assembly of these Trp proteins into homo- or heterotetramers or association with other regulatory proteins. We examined the properties of endogenous capacitative Ca² entry in PHM1 immortalized human myometrial cells that express endogenous hTrpCs 1, 3, 4, 6, and 7 mRNA and in primary human myocytes. In PHM1 cells, activation of the oxytocin receptor or depletion of intracellular Ca² stores with the endoplasmic reticulum calcium pump-inhibitor thapsigargin induced capacitative Ca² entry, which was inhibited both by SKF 96365 and gadolinium (Gd³ ). Whereas unstimulated cells did not exhibit Sr² entry, oxytocin and thapsigargin enhanced Sr² entry that was also inhibited by SKF 96365 and Gd³ . In contrast, Ba² , a poor substrate for Ca² pumps, accumulated in these cells in the absence of the capacitative entry stimulus and also after oxytocin and thapsigargin treatment. Both types of entry were markedly decreased by SKF 96365 and Gd³ . The membrane-permeant derivative of diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), elicited oscillatory increases in PHM1 intracellular Ca² that were dependent on extracellular Ca² . These properties were also observed in primary human myocytes. Overexpression of hTrpC3 in PHM1 cells enhanced thapsigargin-, oxytocin-, and OAG-induced Ca² entry. These data are consistent with the expression of endogenous hTrpC activity in myometrium. Capacitative Ca² entry can potentially contribute to Ca² dynamics controlling uterine smooth muscle contractile activity.

Fer kinase is a 94-kDa cytoplasmic cell-cell actin-based adherens junction (AJ)-associated nonreceptor protein tyrosine kinase (PTK) found in multiple epithelia including the testis, whereas FerT kinase (51 kDa) is the truncated testis-specific form of Fer kinase, lacking the Fps/Fes/Fer/CIP4 (products of oncogenes identified in avian and feline sarcoma, encoding tyrosine protein kinases) and the three coiled-coil domains versus Fer kinase. Yet the role(s) of Fer kinase in AJ dynamics in the testis remains largely unexplored. We have used an in vitro model of AJ assembly with Sertoli-germ cell cocultures and an in vivo model of AJ disassembly in which adult rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) to study changes in the expression and/or localization of Fer kinase during AJ restructuring. Fer kinase/FerT was expressed by Sertoli and germ cells when cultured in vitro. Using an antibody prepared against a synthetic peptide, NH₂-SAPQNCPEEIFTIMMKCWDYK-COOH, corresponding to residues 779–799 of Fer kinase in the rat, which failed to cross-react with FerT kinase, for immunohistochemistry, Fer kinase was detected in the seminiferous epithelium in virtually all stages of the epithelial cycle. At stages XIII-VI, Fer kinase was associated largely with round and elongating spermatids. At stages VII-VIII, Fer kinase associated almost exclusively with round spermatids with very weak staining associated with elongated spermatids. This stage-specific localization of Fer kinase in the epithelium was confirmed by using staged tubules for semiquantitative reverse transcription-polymerase chain reaction. Studies by immunoprecipitation revealed that Fer kinase associated with N-cadherin, γ-catenin, p120^ctn, c-Src (a putative PTK and the product of the transforming, sarcoma-inducing gene of Rous sarcoma virus), Rab 8 (a GTPase), actin, vimentin, but not E-cadherin, afadin, nectin-3, and integrin β1, suggesting Fer kinase associates not only with the actin-based cell-cell AJ structures, such as the N-cadherin/catenin complex (but not the α6β1 integrin/laminin and the afadin/nectin complex), but also with intermediate filament-based cell-cell desmosomes. An induction in Fer kinase expression was detected during Sertoli-germ cell AJ assembly in vitro but not during AF-2364-induced AJ disruption in vivo. Yet this AF-2364-induced Fer kinase plummeting associated with an induction in N-cadherin, β-catenin, and p120^ctn, particularly at the base of the seminiferous epithelium. In summary, Fer kinase structurally associates with the N-cadherin/catenin protein complex in the testis and can possibly be used to mediate signaling function via the cadherin/catenin protein complex.

The model teleost medaka (Oryzias latipes, d-rR.YHNI strain) was used to produce offspring of a defined sex (monosex populations) by crossing experimentally produced YY and XX males to normal females. These monosex populations had the predicted chromosomal constitution as shown by a sex chromosome-specific DNA sequence. However, in XX populations the spontaneous development of males without previous exposure to androgens was observed. Differences in the percentage of male offspring from individual XX breeding pairs indicate a possible variation of unknown genetic factors to be responsible for the development of XX males. The expression of two gonadal genes that are involved in sex differentiation, Dmrt1b(Y) and Fig1a (factor in the germ line α), was analyzed in monosex populations. Dmrt1b(Y) expression correlated strictly with the genotype but not the sexual phenotype. When XY juvenile fish were exposed to 17α-ethynylestradiol at concentrations that induce sex reversal, Dmrt1b(Y) expression was not repressed. However, Dmrt1b(Y) was expressed in XY or YY gonads regardless of the sex and could not be detected in XX individuals. In contrast, the expression of Fig1a correlated with the phenotypic sex: Fig1a was expressed in male juvenile fish exposed to 17α-ethynylestradiol and repressed in fish exposed to 17α-methyltestosterone. The Dmrt1b(Y) expression appears to reflect an early and important event in sex determination and lends support to the suggested key regulatory role of the Dmrt1b(Y) gene in sex determination. This process is apparently hormone insensitive, and the expression of further downstream acting genes can be regulated (directly or indirectly) by sex steroids.

The arginine vasopressin (AVP) type 1a receptor (V1a) is well known to mediate vasoconstriction. In pregnancy, blood flow in the placenta is crucial for sustaining normal growth and development of the fetus. This is the first AVP receptor study in the placenta and fetal membranes. The aim was to compare, quantitatively, the level of V1a gene expression with that of a known marker for vascularization, aquaporin 1 (AQP1). V1a and AQP1 gene expression did not correlate; placental V1a mRNA levels were significantly upregulated at 45 and 66 ± 1 compared with 27, 100 ± 4, and 140 days (term ∼150 days). V1a mRNA levels were much lower in fetal membranes in which no significant difference across gestation was observed. In situ hybridization histochemistry localized V1a gene expression in the maternal component of the placenta similar to the receptor-binding studies using ¹²⁵I-labeled [d(CH₂)₅, sarcosine⁷] vasopressin. No AVP gene expression was observed in the placenta and fetal membranes, which eliminates local AVP production. This increase in V1a expression at 45 and 66 ± 1 days of gestation correlates with the period of maximal placental growth in the sheep and suggests that AVP and V1a receptors may play a hitherto unrecognized role in placental growth, differentiation, and/or function, particularly in the deleterious effects of heat stress, early in pregnancy, on fetal growth.

The contraceptive properties of a gel formulation containing sodium lauryl sulfate were investigated in both in vitro and in vivo models. Results showed that sodium lauryl sulfate inhibited, in a concentration-dependent manner, the activity of sheep testicular hyaluronidase. Sodium lauryl sulfate also completely inhibited human sperm motility as evaluated by the 30-sec Sander-Cramer test. The acid-buffering capacity of gel formulations containing sodium lauryl sulfate increased with the molarity of the citrate buffers used for their preparations. Furthermore, experiments in which semen was mixed with undiluted gel formulations in different proportions confirmed their physiologically relevant buffering capacity. Intravaginal application of the gel formulation containing sodium lauryl sulfate to rabbits before their artificial insemination with freshly ejaculated semen completely prevented egg fertilization. The gel formulation containing sodium lauryl sulfate was fully compatible with nonlubricated latex condoms. Taken together, these results suggest that the gel formulation containing sodium lauryl sulfate could represent a potential candidate for use as a topical vaginal spermicidal formulation to provide fertility control in women.

Bovine luteal cells express class I and II major histocompatibility complex molecules and stimulate T lymphocyte proliferation in vitro. Proliferation of T lymphocytes is greater in cocultures of luteal cells and T lymphocytes collected following administration of a luteolytic dose of prostaglandin (PG) F_2α to the cow. Whether this results from changes in luteal cells that increase their ability to stimulate T lymphocyte proliferation or from changes in T lymphocytes that enhance their ability to respond to luteal cells is unclear. To determine which is the case, luteal cell-T lymphocyte cocultures were performed using luteal cells and T lymphocytes isolated from the same animals before and 8 h after administration of PGF_2α. In the presence of T lymphocytes collected before PGF_2α administration, luteal cells isolated after PGF_2α were more potent stimulators of T lymphocyte proliferation than were luteal cells collected before PGF_2α (P < 0.05). The effect of progesterone on luteal cell-stimulated T lymphocyte proliferation was also evaluated. Proliferation of T lymphocytes was greater (P < 0.05) in cultures containing the cytochrome P450 side-chain cleavage enzyme-inhibitor aminoglutethimide. Exogenous progesterone caused a dose-dependent inhibition of luteal cell-stimulated T lymphocyte proliferation (P < 0.05). Progesterone-receptor mRNA was undetectable in peripheral blood mononuclear cells collected before and after PGF_2α administration, indicating that the effect of progesterone was not mediated via progesterone receptors in lymphocytes. These results imply that specific changes in luteal cells in response to PGF_2α enhance the ability of these cells to stimulate T lymphocyte proliferation. These results also demonstrate that progesterone can suppress luteal cell-stimulated T lymphocyte proliferation.

Stem cells in the male germ line (spermatogonial stem cells [SSCs]) are an important target for male fertility restoration and germ line gene modification. To establish a model system to study the biology and the applications of SSCs in mice, I used a sequential transplantation strategy to analyze the process by which SSCs colonize the stem cell niche after transplantation and to determine the efficiency of the process (homing efficiency). I further analyzed the proliferation kinetics of SSCs after colonization. The number of SSCs gradually decreased during the homing process, and only 12% of SSCs successfully colonized the niche on Day 7 after transplantation, but the number of SSCs increased by Day 14. Thus, homing efficiency of adult mouse SSCs is 12%. These results indicate that SSCs are rapidly lost upon transplantation and require ∼1 wk to settle into their niches before initiating expansion. Using this SSC homing efficiency, I calculated that ∼3000 SSCs exist in one normal adult testis, representing ∼0.01% of total testis cells. Between 7 days and 1 mo after transplantation, SSCs proliferated 7.5-fold. However, they did not significantly proliferate thereafter until 2 mo, and only 8 SSCs supported one colony of donor-derived spermatogenesis from 1 to 2 mo. These results suggest that self-renewal and differentiation of SSCs are strictly regulated in coordination with the progress of an entire unit of regenerating spermatogenesis.

Postnatal development of the ovine uterus between birth and Postnatal Day (PND) 56 involves differentiation of the endometrial glandular epithelium from the luminal epithelium followed by tubulogenesis and branching morphogenesis. These critical events coincide with expression of estrogen receptor α (ERα) by nascent endometrial glands and stroma. To test the working hypothesis that estrogen and uterine ERα regulate uterine growth and endometrial gland morphogenesis in the neonatal ewe, ewes were treated daily from birth (PND 0) to PND 55 with 1) saline and corn oil as a vehicle control (CX), 2) estradiol-17β (E₂) valerate (EV), an ERα agonist, 3) EM-800, an ERα antagonist, or 4) CGS 20267, a nonsteroidal aromatase inhibitor. On PND 14, ewes were hemihysterectomized, and the ipsilateral oviduct and ovary were removed. The remaining uterine horn, oviduct, and ovary were removed on PND 56. Treatment with CGS 20267 decreased plasma E₂ levels, whereas EM-800 had no effect compared with CX ewes. Uterine horn weight and length were not affected by EM-800 or CGS 20267 but were decreased in EV ewes on PND 56. On PND 14 and PND 56, treatment with EV decreased endometrial thickness but increased myometrial thickness. The numbers of ductal gland invaginations and endometrial glands were not affected by CGS but were lower in EM-800 ewes on PND 56. Exposure to EV completely inhibited endometrial gland development and induced luminal epithelial hypertrophy but did not alter uterine cell proliferation. Exposure to EV substantially decreased expression of ERα, insulin-like growth factor (IGF) I, and IGF-II in the endometrium. Results indicate that circulating E₂ does not regulate endometrial gland differentiation or development. Although ERα does not regulate initial differentiation of the endometrial glandular epithelium, results indicate that ERα does regulate, in part, coiling and branching morphogenesis of endometrial glands in the neonatal ewe. Ablation of endometrial gland genesis by EV indicates that postnatal uterine development is extremely sensitive to the detrimental effects of inappropriate steroid exposure.

Chorioamnionitis is a common cause of premature birth and is associated with significant morbidity and mortality in the mother and infant. Preterm birth shares similarities with rejection of the fetal allograft, which is characterized by increased apoptosis of placental trophoblasts. We hypothesized that there is increased trophoblast apoptosis in chorioamnionitis and that this increased apoptosis is mediated by the Fas ligand (FasL)/Fas pathway. To test our hypothesis, we examined placental villous tissues from patients with chorioamnionitis and used the TUNEL assay to demonstrate enhanced trophoblast apoptosis in patients with chorioamnionitis. When the same samples were stained for Fas, there was increased trophoblast Fas expression in patients with chorioamnionitis. To define the mechanisms responsible for this increase in trophoblast apoptosis, we cultured villous explants from uncomplicated term placentas with proinflammatory cytokines and demonstrated a marked increase in trophoblast apoptosis. By blocking FasL, we reduced tumor necrosis factor α-induced and interferon γ-induced apoptosis. These data suggest that chorioamnionitis is associated with increased trophoblast apoptosis and enhanced trophoblast Fas expression. As a complement to our in vivo study, we demonstrated that cytokine-induced trophoblast apoptosis is mediated in part by the FasL/Fas pathway, suggesting that cytokines promote sensitivity to Fas-mediated apoptosis. These mechanisms may be important in perpetuating inflammation in the placental microenvironment and may contribute to the pathogenesis of chorioamnionitis.

Expression of growth differentiation factor 9 (GDF-9), an apparent regulator of follicular development, reportedly differs between compartments of the rodent (oocytes) and human (oocytes and granulosa cells) ovary. To further characterize GDF-9 expression and action in the primate periovulatory follicle, adult female rhesus monkeys received recombinant human gonadotropins to promote multiple follicular development. Whole ovaries or follicular aspirates were obtained before and at various times after administration of an ovulatory dose of hCG; time points for tissue collection spanned the 40-h periovulatory interval. GDF-9 mRNA was detected by reverse transcription polymerase chain reaction assay in each oocyte and every granulosa cell sample examined, but granulosa cell GDF-9 mRNA levels did not change across the periovulatory interval. GDF-9 was also detected in follicular fluid using Western blotting; GDF-9 protein concentration in follicular fluid did not change across the periovulatory interval. Immunocytochemical staining for GDF-9 indicated that oocytes of both small and large antral follicles were positive for GDF-9. GDF-9 immunoreactivity was also present in cumulus granulosa cells and mural granulosa cells near the cumulus stalk. When granulosa cells from preovulatory follicles were exposed to recombinant GDF-9 in culture, GDF-9 increased vascular endothelial growth factor levels in culture medium. These data demonstrate that the cells of the primate periovulatory follicle both produce and respond to GDF-9. However, GDF-9 expression and action differ between rodent and primate follicles, suggesting a possible regulatory role for GDF-9 that is unique to the primate follicle.