bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of the role of nitric oxide synthase 2 in pregnancy using mutant mice
Authors: Burnett TG , Tash JS , Hunt JS
Published: March 2004

Nitric oxide (NO) has been implicated as a signalling molecule in many cellular processes. As nitric oxide synthase 2 (NOS-2) is the main isoform expressed in mouse decidua and metrial gland, mice with a targeted disruption of the gene encoding NOS-2 were used to determine the potential roles of this enzyme during pregnancy. Reproductive success and the morphology of implantation sites throughout pregnancy were compared in NOS-2 deficient (NOS-2-/-) and wild-type (WT) mice. Although there were no significant differences in the duration of gestation or birth weight, NOS-2-/- mice had significantly fewer viable embryos at mid-gestation and delivered smaller litters than did WT mice. Histological sections of uteroplacental units from WT and NOS-2-/- mice were compared to establish the mechanisms underlying the loss of fetuses. No morphological differences were observed on day 6 or day 8 of gestation, indicating that implantation and early development of implantation sites were unaffected by the absence of NOS-2. However, by mid-gestation, decidua of NOS-2-/- mice had reduced cellularity and their decidual arteries had abnormally thickened walls. These observations were quantified by morphometric measurements, which showed a significant reduction in decidual cellular area and a significant increase in the blood vessel wall:lumen ratio in NOS-2-/- mice. The increase in the thickness of the blood vessel walls was not due to abnormal cellular infiltration or to altered expression of alpha-actin in vascular smooth muscle. These results indicate that NOS-2 has a functional role in the maintenance of decidual cellular integrity and development of appropriate uterine vasculature, and may play a supportive role in promoting embryo survival.
Alleviation of the two-cell block of ICR mouse embryos by polyaminocarboxylate metal chelators
Authors: Matsukawa T , Ikeda S , Imai H , Yamada M
Published: March 2004

The present study was undertaken to examine the effects of various transition metal ion chelators, both polyaminocarboxylates (including nitrilotriacetate (NTA), ethylenediaminediacetate (EDDA), ethyleneglycolbistetraacetate (EGTA), ethylenediaminetetraacetate (EDTA) and diethylenetriaminepentaacetate (DTPA)) and non-polyaminocarboxylates (dipicolinic acid and deferoxamine), on the development in vitro of one-cell ICR strain mouse embryos to the four-cell and blastocyst stages. The order of stability constants of polyaminocarboxylates for transition metal ions such as zinc, copper and iron is as follows: NTA < or = EDDA < EGTA < EDTA < DTPA. Addition of 10 or 100 micromol polyaminocarboxylates x l(-1) to the medium significantly enhanced the development of most one-cell embryos (66-88%) beyond the two-cell stage compared with that (< 25%) in medium without polyaminocarboxylates. Although EDDA, EDTA and DTPA at 10 micromol x l(-1) induced the development of most one-cell embryos to the four-cell stage and beyond, a higher concentration (100 micromol x l(-1)) of NTA and EGTA was required to obtain a similar result. Therefore, the ability of polyaminocarboxylates to overcome the two-cell block is not correlated with their potency to chelate transition metal ions. In contrast, the non-polyaminocarboxylates dipicolinic acid and deferoxamine, at 10 and 100 micromol x l(-1), did not have the same effect. Taken together, the results indicate that the ability of polyaminocarboxylates to overcome the two-cell block in embryo development is due to some common feature or features other than the ability to chelate transition metal ions.
Effects of oestrogen treatment on testicular descent, inguinal closure and prostatic development in a male marsupial, Macropus eugenii
Authors: Coveney D , Shaw G , Renfree MB
Published: March 2004

This study reports the effect of oestrogen treatment on the development of the genital ducts, prostate gland, testicular descent and inguinal canal closure in male tammar wallaby young treated with oestrogen over four time spans during the first 25 days of pouch life (days 0-10, 10-15, 15-25 and 0-25) and sampled at day 50. In control males, the Mullerian ducts had regressed and the Wolffian ducts had developed into the vas deferens and epididymis. The prostate gland had formed epithelial buds extending from the ventral, lateral and posterior walls of the urethra. The testes were in the neck of the scrotum and the gubernaculum and processus vaginalis were present at the base of the scrotum. In most males treated with oestradiol from day 0 to day 25, the testes had failed to descend by day 50. The gubernaculae were long and thin. The retained Mullerian ducts formed a lateral vaginal expansion like that of normal day 50 females. The Wolffian ducts of the males treated on days 0-25 were regressed, but were present in males in the other three treatment groups. The prostate glands were hyperplastic and epithelial budding was highly invasive. Some treated males from the day 10-25 and 0-25 groups had inguinal hernias. These results demonstrate that oestrogen treatment has profound effects on the development of the internal genitalia of a male marsupial, preventing inguinal closure and interfering with testicular descent. Therefore, the tammar wallaby may provide a useful experimental model animal in which to investigate the hormonal control of testicular migration and closure of the inguinal canal.
A quantitative (stereological) study of the effects of experimental unilateral cryptorchidism and subsequent orchiopexy on spermatogenesis in adult rabbit testis
Authors: Zhang RD , Wen XH , Kong LS , Deng XZ , Peng B , Huang AP , Wan Y , Yang ZW
Published: March 2004

The aim of this study was to examine the controversial effects of experimental unilateral cryptorchidism and subsequent orchiopexy on the number of germ cells and other morphometric characteristics of testicular and epididymal structures in adult rabbits. Unilateral cryptorchidism was induced in 11 mature male New Zealand white rabbits by returning one testis, together with the ipsilateral epididymis, to the abdominal cavity via a surgical procedure. After 3 months, testes and epididymides were removed from six animals (and from six age-matched control animals that did not undergo the surgery). Orchiopexy was performed on the five remaining animals and the testes and epididymides of these animals (and an additional six age-matched control animals) were removed 7 weeks later. A contemporary, unbiased and efficient stereological tool, the optical disector, was used to estimate the number of nuclei in the testis and epididymis using methacrylate-embedded sections of 25 micron in thickness. Cryptorchidism resulted in severe testicular atrophy and spermatogenic arrest: type A spermatogonia and Sertoli cells only were seen in the seminiferous epithelium, and the number of type A spermatogonia per testis was reduced by 84%. After orchiopexy, the testis remained atrophied and the number of type A spermatogonia returned to the near-normal range in four of five animals, but spermatogenesis was recovered only partially at the stage of early primary spermatocytes (one animal), late primary spermatocytes (two animals) or spermatids (one animal). In conclusion, cryptorchidism caused severe spermatogenic arrest that was potentially recoverable (in view of the restoration of the number of type A spermatogonia), but orchiopexy failed to induce full recovery of spermatogenesis.
Effect of local heating of rat testes after suppression of spermatogenesis by pretreatment with a GnRH agonist and an anti-androgen
Authors: Setchell BP , Ploen L , Ritzen EM
Published: March 2004

The effects of local heating of rat testes, in which spermatogenesis had been suppressed with injections of a GnRH agonist and an anti-androgen, were examined. Although the detrimental effects of heating were not as marked as those found in the testes of non-injected rats, the testes in which spermatogenesis was suppressed also showed a significant reduction in mass, the number of spermatozoa, tubular diameter and the percentage of normal tubular cross-sections at day 35 after heating. The results indicate that heating has an effect on cells in the testis other than those shown to be most susceptible to heat, namely pachytene spermatocytes and early spermatids, which were absent or markedly reduced in number when spermatogenesis was suppressed. The long-term effects of heating on the above parameters, as reported in a previous study, were also confirmed. However, in testes in which spermatogenesis was suppressed at the time of heating, there appeared to be no or a reduced long-term impairment of spermatogenesis, as determined by testis mass, the percentage of qualitatively normal tubules and epididymal sperm counts.
Anti-proliferative effects of progesterone antagonists in the primate endometrium: a potential role for the androgen receptor
Authors: Brenner RM , Slayden OD , Critchley HO
Published: March 2004

In women and non-human primates, treatment with anti-progestins suppresses oestrogen-dependent mitotic activity in the endometrial glands. This anti-proliferative effect is paradoxical, because anti-progestins do not bind to the oestrogen receptor. Although this phenomenon has been termed a 'non-competitive anti-oestrogenic effect', it does not occur in all species or in other regions of the primate reproductive tract, so is best referred to as an 'endometrial anti-proliferative effect'. The abundance of androgen receptors is greatly increased by anti-progestin treatment, especially in the glandular epithelium in non-human primates and women. Such an increase could lead to an enhancement of androgen action in the endometrium. As androgens suppress oestrogen-dependent endometrial proliferation, the increased abundance of androgen receptors could mediate the anti-proliferative effects of anti-progestin treatment. This brief review evaluates the implications of these findings.
Developmental potential of cloned mouse embryos reconstructed by a conventional technique of nuclear injection
Authors: Rybouchkin A , Heindryckx B , Van der Elst J , Dhont M
Published: March 2004

The fact that most of the advances in mouse cloning by nuclear transfer originate from research in a limited number of laboratories demonstrates the complexity of the reported technologies. The development of alternative and more simple techniques of nuclear transfer may therefore be of interest. Furthermore, the preimplantation biology of cloned mouse embryos originating from somatic cells has not yet been studied in detail. In the present study, a modified conventional injection (mCI) technique for cloning mice from somatic cells is described. The preimplantation development and morphology of the resulting nuclear transfer embryos in comparison with parthenogenetic embryos and embryos obtained by intracytoplasmic sperm injection (ICSI) under comparable conditions was also studied. Finally, the capacity of nuclear transfer embryos for full-term development was investigated. Eighty-nine per cent of oocytes injected with cumulus cell nuclei under mCI conditions survived and formed zygotes. However, the rate of development of these zygotes to the blastocyst stage was significantly lower (29%) than that of ICSI or parthenogenetic zygotes (95 and 92%, respectively). Cloned blastocysts had a significantly lower mean number of cells in the inner cell mass (9) and trophectoderm (52) and a lower inner cell mass:total cell ratio (14%) than did their counterparts (31, 143 and 18% for ICSI and 21, 92 and 18% for parthenogenetic blastocysts, respectively). This correlated with a significantly higher proportion of dead cells in the cloned blastocysts. The poor quality of cloned blastocysts may explain the low rate of full-term fetal development of somatic mouse clones.
A potential molecular mechanism for regulating pre-mRNA splicing of implantation-related genes through unique uterine expression of splicing factor SC35 in women and rhesus monkeys
Authors: Nie GY , Hampton AL , Fu GQ , Liu YX , Findlay JK , Salamonsen LA
Published: March 2004

Splicing factor SC35 is an essential component of the spliceosome, the cellular apparatus that removes introns from pre-mRNA to provide alternatively spliced isoforms. Many proteins associated with development of uterine receptivity and embryo implantation are present as isoforms, the tissue-specific expression of which may be regulated through alternative splicing. SC35 was identified as being increased at implantation sites during early pregnancy in mice. However, the present study has demonstrated that SC35 is present in human and rhesus monkey endometrium, that the protein is increased during the secretory phase of the oestrous cycle compared with the proliferative phase in both these primates and that it is present in a distinct pattern within the nucleus of both epithelial and stromal cells, as well as in cells of the vasculature. Both the intensity of immunoreactive protein and the proportion of cells that stain for SC35 alter with the phase of the oestrous cycle. A very precise expression pattern of SC35 (both protein and mRNA) was seen during early placentation in rhesus monkeys. At implantation sites between day 24 and day 35 of early pregnancy, SC35 was expressed strongly in cytotrophoblasts within the trophoblastic shell, in syncytiotrophoblast at the periphery of the cell column and in both cytotrophoblast and syncytiotrophoblast in the floating villi. In the adjacent maternal decidua, expression of SC35 was weak. These results indicate a role for SC35 in preparation of a receptive uterus, in the provision of secreted proteins to support blastocyst development and in trophoblast invasion.
Basigin expression and hormonal regulation in the rat uterus during the peri-implantation period
Authors: Xiao LJ , Diao HL , Ma XH , Ding NZ , Kadomatsu K , Muramatsu T , Yang ZM
Published: March 2004

Basigin is essential for fertilization and implantation. The aim of this study was to determine the expression and hormonal regulation of the basigin gene in the rat uterus during the peri-implantation period. Basigin mRNA was localized strongly in the luminal epithelium on day 1 of pregnancy and gradually decreased to a basal concentration from day 3 to day 5 of pregnancy. Basigin mRNA and protein were expressed strongly in the implanting blastocyst and primary decidua on day 6 of pregnancy. A similar expression pattern was also induced in the uterus after delayed implantation was terminated by oestrogen treatment and the embryo implanted, whereas expression was not detected during delayed implantation. Basigin expression was not detected on day 6 of pseudopregnancy. Basigin mRNA was expressed strongly in the decidua on days 7 and 8 of pregnancy. Furthermore, both basigin mRNA and protein were induced in the decidua during artificial decidualization. In addition, oestrogen stimulated strong expression of basigin mRNA in the uterine epithelium of ovariectomized rats. These findings indicate that basigin may play a role during implantation and decidualization in rats.
Characterization of the coupling activity for the binding of inter-alpha-trypsin inhibitor to hyaluronan in human and bovine follicular fluid
Authors: ODum L , Andersen CY , Jessen TE
Published: March 2004

The plasma proteinase inter-alpha-trypsin inhibitor is necessary for normal expansion of the cumulus-oocyte complex (COC) and lack of inter-alpha-trypsin inhibitor results in severe infertility. After diffusion from the circulation into the follicles, inter-alpha-trypsin inhibitor is incorporated into the extracellular hyaluronan network of the expanding COC. However, mixing isolated inter-alpha-trypsin inhibitor with hyaluronan in vitro does not result in coupling to hyaluronan. Other components must be present. A recently developed electrophoretic technique by which hyaluronan-bound inter-alpha-trypsin inhibitor is immobilized was used to demonstrate coupling activity in human and bovine follicular fluid that is necessary for the formation of a firm binding between inter-alpha-trypsin inhibitor heavy chains and hyaluronan, as observed in vivo. No coupling activity could be detected in human serum. Coupling occurred only in the presence of follicular fluid. The coupling activity of follicular fluid was irreversibly destroyed by heat treatment, lowering of pH or tryptic digestion, indicating that the coupling activity is associated with a protein. Calcium ions are essential for the coupling reaction. The binding reaction in vitro using intact inter-alpha-trypsin inhibitor is slow and occurs over 24 h. The early-formed complexes between inter-alpha-trypsin inhibitor and hyaluronan contain small amounts of bikunin, whereas the end product contains heavy chains and essentially no bikunin. The heavy chains released from inter-alpha-trypsin inhibitor by NaOH treatment bound immediately to hyaluronan, indicating that the dissociation of heavy chains from inter-alpha-trypsin inhibitor is the rate-limiting step. In conclusion, at least four components are essential for the covalent binding of heavy chains to hyaluronan: inter-alpha-trypsin inhibitor and calcium from plasma, hyaluronan and one or more proteins found in follicular fluid.
Follistatin concentrations in maternal and fetal fluids during the oestrous cycle, gestation and parturition in Merino sheep
Authors: McFarlane  , Xia Y , O'Shea T , Hayward S , O'Connor AE , De Kretser DM
Published: March 2004

The aim of this study was to investigate the changes in follistatin, an activin binding protein, during the oestrous cycle, gestation and parturition in ewes using a radioimmunoassay for total follistatin, which uses dissociating reagents to remove the interference of activin. Follistatin concentrations remained unchanged (2.7 +/- 0.2 ng ml(-1)) during the oestrous cycle and decreased as pregnancy progressed. Follistatin concentrations in allantoic fluid also decreased during gestation, whereas in amniotic fluid follistatin concentrations reached a peak at day 75 of gestation (9.8 ng ml(-1)) and had decreased to 4.4 ng ml(-1) at day 140. Follistatin concentrations in fetal blood (7.0 +/- 0.5 ng ml(-1)) did not change from day 50 to day 140 of gestation but were significantly higher than in matched maternal samples (3.1 +/- 0.3 ng ml(-1)). Circulating follistatin in ewes was significantly increased on the day of parturition (5.6 +/- 0.6 ng ml(-1)) compared with the days before parturition (2.7 +/- 0.4 ng ml(-1)), but had decreased by day 2 after birth. Blood samples from newborn lambs showed that plasma follistatin concentration (13.4 +/- 2.3 ng ml(-1)) was significantly higher than that of the mothers and remained high for at least 7 days after birth. These data support previous studies of the human menstrual cycle indicating that follistatin is not an endocrine signal from the ovary; however, in contrast to human pregnancies, follistatin concentrations in sheep decreased and become high only after or during parturition. This difference observed between species may reflect different physiological effects of follistatin or may be the result of measurement of different isoforms.
The cooperative action of angiotensin II with subluteolytic administration of PGF2alpha in inducing luteolysis and oestrus in the cow
Authors: Hayashi K , Tanaka J , Hayashi KG , Hayashi M , Ohtani M , Miyamoto A
Published: March 2004

There is evidence that the potent vasoconstrictor angiotensin II (Ang II) regulates luteal functions. In the present study, the effect of an intraluteal injection of Ang II alone or in combination with pretreatment with a subluteolytic i.m. dose of PGF(2alpha) on the concentration of plasma progesterone, corpus luteum regression and duration of the oestrous cycle was investigated. Cows were assigned randomly to receive an intraluteal injection of either: (i) 500 microl saline 30 min after i.m. administration of saline (control, n = 7); (ii) 500 microl saline 30 min after i.m. administration of a subluteolytic dose (125 microg) of a PGF(2alpha) analogue (1/4 PGF(2alpha), n = 5); (iii) 2 mg Ang II in 500 microl saline 30 min after i.m. administration of saline (Ang II, n = 5); or (iv) 2 mg Ang II in 500 microl saline 30 min after i.m. administration of a subluteolytic dose (125 microg) of the PGF(2alpha) analogue (1/4 PGF(2alpha)/Ang II, n = 6) on day 12 of the oestrous cycle. There were no significant changes in plasma progesterone concentrations in the control, 1/4 PGF(2alpha) or Ang II treatment groups. Treatment with 1/4 PGF(2alpha)/Ang II decreased plasma progesterone concentration, and induced luteolysis and oestrus. The onset of oestrus in cows treated with full-dose (500 microg) PGF(2alpha) (3.1 +/- 0.2 (mean +/- SEM) days after treatment) was significantly earlier than that in cows treated with 1/4 PGF(2alpha)/Ang II (4.8 +/- 0.2 days after treatment) (P < 0.05). The results from the present study demonstrate that an intraluteal injection of Ang II after i.m. administration of a subluteolytic dose of PGF(2alpha) analogue induces luteolysis and oestrus. Thus, these results support the contention that Ang II is directly correlated with the process of luteal regression in cows.
Spermatogenesis and steroidogenesis in mouse, hamster and monkey testicular tissue after cryopreservation and heterotopic grafting to castrated hosts
Authors: Schlatt S , Kim SS , Gosden R
Published: March 2004

Retrieval, extracorporal storage and autotransplantation of testicular tissue could become an important strategy for preserving male gonadal function. The present study used syngeneic and immunodeficient nude mice as hosts, and immature and adult mice, neonatal and adult photoregressed Djungarian hamsters and neonatal marmosets to identify the potential of testicular tissue grafting to maintain the morphological and functional integrity of the testis. Testicular tissue was grafted s.c. either as fresh tissue or after cryopreservation into adult, orchidectomized hosts. The mice that received rodent testis tissue were autopsied 50 days later, and blood samples were collected. Sixty-five per cent of mouse isografts contained morphologically normal testicular tissue and seminiferous tubules with some degree of spermatogenic recovery. Mature spermatozoa were recovered after enzymatic disaggregation. Although the recovery of spermatogenesis was limited in adult mouse and hamster tissue, complete spermatogenesis was observed in grafts from immature rodents. Testicular tissue from neonatal marmosets developed up to the stage of spermatocytes at day 135 after xenografting. Androgen concentrations were comparable in intact control mice and in mice receiving fresh mouse and hamster grafts, slightly lower in mice receiving cryopreserved grafts and adult photoregressed hamster tissue, and low in castrated control mice and in mice receiving marmoset tissue. These results show that isografts and xenografts of immature and adult testicular tissue become functionally active as a s.c. graft in the mouse and that this approach might be useful in combination with cryopreservation as a tool for storage and activation of the male germ line and androgen replacement therapy in patients.
Caspase activity and expression of cell death genes during development of human preimplantation embryos
Authors: Spanos S , Rice S , Karagiannis P , Taylor D , Becker DL , Winston RM , Hardy K
Published: March 2004

It has been observed that apoptosis occurs in human blastocysts. In other types of cell, the characteristic morphological changes seen in apoptotic cells are executed by caspases, which are regulated by the BCL-2 family of proteins. This study investigated whether these components of the apoptotic cascade are present throughout human preimplantation development. Developing and arrested two pronucleate embryos at all stages were incubated with a fluorescently tagged caspase inhibitor that binds only to active caspases, fixed, counterstained with 4,6-diamidino-2-phenylindole (DAPI) to assess nuclear morphology and examined using confocal microscopy. Active caspases were detected only after compaction, at the morula and blastocyst stages, and were frequently associated with apoptotic nuclei. Occasional labelling was seen in arrested embryos. Expression of proapoptotic BAX and BAD and anti-apoptotic BCL-2 was examined in single embryos using RT-PCR and immunohistochemistry. BAX and BCL-2 mRNAs were expressed throughout development, whereas BAD mRNA was expressed mainly after compaction. Simultaneous expression of BAX and BCL-2 proteins within individual embryos was confirmed using immunohistochemistry. The onset of caspase activity and BAD expression after compaction correlates with the previously reported appearance of apoptotic nuclei. As in other types of cell, human embryos express common molecular components of the apoptotic cascade, although apoptosis appears to be suppressed before compaction and differentiation.
Effects of oocyte maturation regimen on the relative abundance of gene transcripts in bovine blastocysts derived in vitro or in vivo
Authors: Knijn HM , Wrenzycki C , Hendriksen PJ , Vos PL , Herrmann D , van der Weijden GC , Niemann H , Dieleman SJ
Published: March 2004

Bovine embryos produced in vitro differ substantially from embryos produced in vivo in the mRNA expression patterns of genes important for development. Several factors in the in vitro production systems have profound effects on embryonic mRNA expression patterns. The effects of the type of maturation on the expression pattern of genes important for development in blastocysts produced in vitro have not yet been investigated. The aim of the present study was to investigate the effects of various maturational protocols on the relative abundance of a panel of six marker genes, indicative of compaction and cavitation, metabolism, stress susceptibility and RNA processing, in bovine blastocysts produced in vitro. Four groups of blastocysts were analysed by a sensitive semi-quantitative RT-PCR assay. Blastocysts were produced in vitro from oocytes of different origin from: (1) 3-8 mm follicles; (2) preovulatory follicles before the LH surge; and (3) preovulatory follicles 24 h after the LH surge. The first two groups were matured in vitro, whereas the third group had undergone maturation in vivo. A fourth group comprised blastocysts developed entirely in vivo. Expression of glucose transporter 1 was significantly (P < 0.05) higher, and expression of desmocollin 2 and plakophilin tended to be higher (P < 0.1) for in vivo (group 4) compared with in vitro blastocysts (group 1), whereas no differences were found for heat shock protein 70.1, E-cadherin and poly(A) polymerase. Expression of the six transcripts did not differ among blastocysts produced in vitro from oocytes of groups 1, 2 and 3. Results indicate that alterations in the relative abundance of these transcripts in blastocysts produced in vitro cannot primarily be attributed to the origin of the oocyte, but are likely to have been induced by post-maturation or fertilization culture conditions.
Quantification of mast cells and microflora in the vaginal cul-de-sac of the brushtail possum (Trichosurus vulpecula)
Authors: Mahoney PM , Hurst PR , McLeod BJ , McConnell MA
Published: March 2004

Mast cells are typically associated with biological responses to intracellular parasitic or bacterial invasion in gut and pulmonary tissues. Large numbers of these cells are present in the vaginal cul-de-sac, a component of the reproductive tract in female brushtail possums (Trichosurus vulpecula). The presence of mast cells in this area may reflect microbial presence or indicate a role for mast cells in reproductive function in this marsupial. In this study, the number and spatial location of mast cells in vaginal cul-de-sac tissues at different reproductive stages was determined, and related to the presence of microflora. Vaginal cul-de-sac tissue was collected from juvenile (n = 5), lactationally anoestrous and cyclic adult brushtail possums (n = 6 per group). Oestrous cyclicity was induced by removal of pouch young and reproductive status was confirmed by laparoscopy. Tissue was collected aseptically for microbiological procedures. The fractionator and optical disector stereological methods were used to quantify mast cell populations. In all groups, microflora populations were low (< 5 x 10(5) organisms g(-1)). Mast cell density in epithelial tissue was highest in lactationally anoestrous and luteal phase brushtail possums and lowest in follicular phase animals. No correlation between the numbers of bacteria isolated and mast cell density was apparent. Gram stained tissue showed no evidence of bacterial invasion. Mast cell density was significantly lower in epithelial tissue from follicular phase animals, corroborating previous studies in which an increase in mast cell degranulation coincided with high oestrogen concentrations.
Interleukin 8 in the cervix of non-pregnant ewes
Authors: Mitchell SE , Robinson JJ , King ME , McKelvey WA , Williams LM
Published: March 2004

Studies of cervical artificial insemination of ewes at hormone-synchronized oestrus indicate that the cervix remains relatively impenetrable to semen, in contrast to naturally breeding animals. During parturition the inflammatory response plays an important part in cervical dilation and possibly, to a lesser extent, in the non-pregnant cervix at oestrus to facilitate the transcervical transport of semen. The expression of interleukin 8 (IL-8), a pro-inflammatory cytokine in the ovine cervix, has been mapped and quantified, using semi-quantitative in situ hybridization, to ascertain the role played by inflammation in the ovine cervix during natural and artificially induced oestrous cycles. IL-8 gene expression was observed in both the luminal epithelium and fibroblastic cells of the cervix. The presence of IL-8 was confirmed using immunohistochemistry. IL-8 gene expression in the luminal epithelium varied throughout the oestrous cycle and was highest at oestrus and at day 5 of the oestrous cycle. In ewes artificially induced to ovulate, either by the withdrawal of progesterone pessaries after treatment for 12 days, or by two i.m. injections of prostaglandin 9 days apart, IL-8 gene expression at oestrus was significantly lower than it was at natural oestrus. Insemination increased IL-8 gene expression in progesterone-synchronized ewes. These data support the hypothesis that IL-8-induced inflammation is important in normal cervical function and that this process is inhibited during artificial synchronization of the oestrous cycle and is increased by exposure to semen.
Expression of prolactin-related protein I at the fetomaternal interface during the implantation period in cows
Authors: Yamada O , Todoroki J , Kizaki K , Takahashi T , Imai K , Patel OV , Schuler LA , Hashizume K
Published: March 2004

The bovine placenta secretes multiple molecules during implantation and placentation, many of which are produced by binucleate cells. In this study, production of prolactin-related protein I (PRP-I), a member of the non-classical prolactin-related family, was investigated during the implantation period in cows. Expression of bovine PRP-I (bPRP-I) in the placentome was examined during the preimplantation (days 17-19), implantation (days 20-25) and post-implantation (days 30-60) periods by immunohistochemistry, immunofluorescence and in situ hybridization. During the preimplantation period, both bPRP-I and bovine placental lactogen (bPL) were undetectable in trophoblastic cells. Both bPRP-I mRNA and protein appeared first at day 20 of gestation in trophoblastic binucleate cells and multinuclear cells that might migrate into the endometrium and fuse to epithelium; however, no bPL was detected in binucleate cells at this time. After implantation, on day 30, both bPRP-I and bPL were detected in binucleate cells and were co-expressed in the same cells. These data indicate that bPRP-I may play a role before implantation and that bPRP-I may be an excellent marker for trophoblastic cell differentiation, as well as a candidate for pregnancy diagnosis.
Vertebrate sex determination: many means to an end
Authors: Morrish BC , Sinclair AH
Published: March 2004

The differentiation of a testis or ovary from a bipotential gonadal primordium is a developmental process common to mammals, birds and reptiles. Since the discovery of SRY, the Y-linked testis-determining gene in mammals, extensive efforts have failed to find its orthologue in other vertebrates, indicating evolutionary plasticity in the switch that triggers sex determination. Several other genes are known to be important for sex determination in mammals, such as SOX9, AMH, WT1, SF1, DAX1 and DMRT1. Analyses of these genes in humans with gonadal dysgenesis, mouse models and using in vitro cell culture assays have revealed that sex determination results from a complex interplay between the genes in this network. All of these genes are conserved in other vertebrates, such as chickens and alligators, and show gonad-specific expression in these species during the period of sex determination. Intriguingly, the sequence, sex specificity and timing of expression of some of these genes during sex determination differ among species. This finding indicates that the interplay between genes in the regulatory network leading to gonad development differs between vertebrates. However, despite this, the development of a testis or ovary from a bipotential gonad is remarkably similar across vertebrates.
Role of low circulating FSH concentrations in controlling the interval to emergence of the subsequent follicular wave in cattle
Authors: Ginther OJ , Bergfelt DR , Beg MA , Kot K
Published: March 2004

The intervals between emergence of follicular waves 1 (first wave of an oestrous cycle) and 2, and between the associated FSH surges (surges 1 and 2), were studied in control (n = 7) and recombinant bovine (rb)FSH-treated (n = 7) heifers. The expected start of the deviation in follicle diameter between the two largest follicles of wave 1 was defined as the day on which the largest follicle reached 8.5 mm (day 0). In the control heifers, circulating concentrations of FSH decreased and oestradiol increased between day 0 and day 1.5 or day 2.0 in a reciprocal relationship. The opposite reciprocal relationship between an FSH increase and an oestradiol decrease occurred during the next 3 days. This temporal result is consistent with a negative systemic effect of oestradiol on FSH at this time. rbFSH was administered in a dosage regimen that was expected to result in a similarity between FSH surge 2 in the rbFSH-treated group and surge 2 in the control group. On average, surge 2 and wave 2 occurred approximately 2 days earlier in the rbFSH-treated group than in the control group, and characteristics of the FSH surge and follicular wave were similar (no significant differences) between groups. These results support the hypothesis that low circulating FSH concentrations after the deviation in follicle diameter control the interval to emergence of the subsequent follicular wave. However, in one of seven rbFSH-treated heifers, the largest follicle from the apparent stimulation of rbFSH reached only 5.7 mm; therefore, the possibility of involvement of additional mechanisms cannot be dismissed.
Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmental potential after vitrification
Authors: Liebermann J , Tucker MJ
Published: March 2004

The present study was undertaken to examine the effect of a cooling regimen during vitrification on survival and morphological appearance of human oocytes after warming and the developmental potential of day 3 embryos. Aged human oocytes that had failed to fertilize and human embryos derived from abnormally fertilized zygotes that showed one pronucleus or three or more pronuclei were used as models in this study. In the first part of the study, 928 aged human oocytes that had failed to fertilize were vitrified. The viability of oocytes after vitrification using the hemi-straw system was slightly higher than it was using the cryoloop after warming (85.4% (410 of 480) versus 80.6% (361 of 448)), but the difference was not significant. In the second part of the study, 266 embryos were vitrified. The survival of day 3 embryos after vitrification was improved by using a mixture of ethylene glycol and dimethyl sulphoxide in combination with the hemi-straw system method rather than the cryoloop method (89.7% (122 of 136) versus 83.8% (109 of 130)), but the difference was again not significant. The potential for development up to compaction on day 4 in embryos vitrified using the hemi-straw system method was significantly higher (37.7%) than it was in embryos vitrified using the cryoloop (29.4%; chi(2), P = 0.002). The hemi-straw system and cryoloop methods of vitrification are both successful, easy to perform, and demonstrate the ability of both carriers to vitrify different stages of development (oocytes and day 3 embryos). In addition, the success of both methods appears to be related to the rate of cooling and the lower concentration of cryoprotectant used.
Regulation of steroid synthesis and apoptosis by insulin-like growth factor I and insulin-like growth factor binding protein 3 in human corpus luteum during the midluteal phase
Authors: Villavicencio A , Iniguez G , Johnson MC , Gabler F , Palomino A , Vega M
Published: March 2004

The aim of the present study was to investigate the action of insulin-like growth factor I (IGF-I) and insulin-like growth factor-binding protein 3 (IGFBP-3) on steroidogenesis and apoptosis in human corpus luteum during the midluteal phase. Slices from corpora lutea were incubated for 4 h with IGF-I or IGFBP-3. Progesterone, oestradiol, androstenedione and testosterone concentrations were determined by radioimmunoassay; caspase 3 expression was assessed by immunohistochemistry; bcl-2, bax and P(450arom) expression were assessed by RT-PCR; and apoptosis was detected by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling. The results showed that addition of IGF-I stimulated progesterone production (150%, P < 0.01), oestradiol production (65%, P < 0.05) and bcl-2 gene expression (approximately 200%, P < 0.05), but decreased apoptosis (P < 0.05). In contrast, IGFBP-3 reduced steroid production and increased bax gene expression and the percentage of apoptotic cells (P < 0.05). Neither IGF-I nor IGFBP-3 had an effect on P(450arom) expression or on the concentrations of its substrates. However, maximum expression of caspase 3 was detected in corpus luteum during the midluteal phase. In conclusion, these results indicate that IGF-I and IGFBP-3 act as regulatory peptides of the function of the human corpus luteum during the midluteal phase. This action may be direct or mediated by steroid production or by bcl-2-bax expression.
Amino acid requirements for maturation of rhesus monkey (Macacca mulatta) oocytes in culture
Authors: Zheng P , Bavister BD , Ji WZ
Published: March 2004

This study evaluated the effects of different amino acid formulations on supporting meiotic and cytoplasmic maturation of rhesus monkey (Macacca mulatta) oocytes in vitro. Five hundred and forty-six cumulus-oocyte complexes (COCs) aspirated from unstimulated adult monkey follicles (> or = 1000 microm in diameter) were cultured in either modified Connaught Medical Research Laboratories 1066 medium (mCMRL-1066) or in one of eight chemically defined media (modified basic medium 5 supplemented with 5.5 mmol glucose l(-1), 0.003 mmol pantothenic acid l(-1) and different amino acid formulations) as below: (1) modified basic medium 5 (mBM5) containing no amino acid; (2) mBM5 + 0.2 mmol glutamine l(-1); (3) mBM5 + 11 amino acids from hamster embryo culture medium 6 (HECM-6) (11 AA); (4) mBM5 + Eagle's non-essential amino acids (NEA); (5) mBM5 + NEA + 0.2 mmol glutamine l(-1); (6) mBM5 + Eagle's essential amino acids (EA) without glutamine; (7) mBM5 + EA + 0.2 mmol glutamine l(-1); (8) mBM5 + Eagle's 20 amino acids (20 AA) + 0.2 mmol glutamine l(-1); and (9) mCMRL-1066 (control). All media contained FSH, LH, oestradiol and progesterone. After maturation, mature oocytes were subjected to the same fertilization and embryo culture procedures. COCs matured in treatment 5 had greater potential to progress to metaphase II (66%; P < 0.05) than did those in treatments 1 (37.3%), 2 (48.3%), 3 (41%), 6 (41%) and 9 (43%). Oocytes matured in treatment 8 had the best morula (53%) and blastocyst (18%) developmental responses (P < 0.05). The lowest (P < 0.05) morula and blastocyst developmental responses were obtained from COCs matured in treatments 1 (0%) and 6 (8%). The other media supported intermediate embryonic development (range 11-38% of morula and blastocyst). These results indicate that the choice of amino acids affects the competence of oocyte maturation and that Eagle's 20 AA with 0.2 mmol glutamine l(-1) is more efficient than the other amino acid formulations for maturation of rhesus monkey oocytes.
Effects of oestradiol and progesterone on secretion of gonadotrophins and health of first wave follicles during the oestrous cycle of beef heifers
Authors: Austin EJ , Mihm M , Evans AC , Ireland JL , Ireland JJ , Roche JF
Published: March 2004

Antral follicle development in cattle is initially FSH dependent and then LH dependent. The aim of the present study was to determine the effects of oestradiol- and progesterone-induced suppression of FSH and LH on growth and differentiation of first wave follicles. Cyclic heifers (n = 45, n = 6-10 per group) received the following i.m. injections or treatments beginning 30 h after oestrus: (i) saline (controls); (ii) 0.75 mg oestradiol benzoate (ODB); (iii) insertion of a progesterone-releasing intravaginal device (PRID) for 42 h (progesterone); (iv) 0.75 mg oestradiol benzoate plus PRID (ODB plus progesterone); (v) 0.75 mg ODB plus injection of 1 mg Ovagen(TM) at 33, 39 and 45 h after onset of oestrus (ODB plus FSH). In Expt 1, follicle development was monitored by ovarian ultrasonography once a day. In Expt 2, heifers were ovariectomized. Emergence of the first follicle wave and dominant follicle selection were delayed in ODB plus progesterone-treated heifers compared with controls. Interval to nadir FSH concentration was shorter in ODB-, progesterone- and ODB plus progesterone-treated heifers compared with controls. Frequency of LH pulses was unaffected in ODB- or ODB plus FSH-treated heifers, decreased in progesterone-treated heifers and further decreased in ODB plus progesterone-treated heifers. Intrafollicular oestradiol concentrations were lower in the largest follicle from ODB plus progesterone-treated heifers compared with control (66 h) heifers, but follicle diameter and concentrations of insulin-like growth factor binding proteins (IGFBPs) and inhibin forms were unaffected. Treatment with ODB decreased follicular oestradiol concentration in smaller follicles in the cohort. It is concluded that growing cohort follicles are uniformly responsive to increased FSH concentration but differentially responsive to suppressed FSH and LH release, which is consistent with an LH-mediated survival advantage of the largest follicle in the cohort before cessation of the growth of remaining follicles in the cohort occurs.
Replacement of nuclear protein by histone in pig sperm nuclei during in vitro fertilization
Authors: Nakazawa Y , Shimada A , Noguchi J , Domeki I , Kaneko H , Kikuchi K
Published: March 2004

Sperm-specific nuclear protamines are dissociated before decondensation of sperm nuclei during fertilization in pigs. In the present study, replacement of nuclear protein by histone in boar spermatozoa during in vitro fertilization was evaluated by immunohistochemistry using anti-histone antibody. First, the specificity of the antibody used in this study was examined. Immunohistochemistry of the testes and epididymides indicated that somatic nuclei, but not elongated spermatids or maturing spermatozoa, were immunoreactive. Furthermore, immunoreaction was diminished after the antibody had been preincubated with unfractionated histone, indicating that the antibody was specific for the somatic nuclear histone. Immunohistochemistry of serial sections of oocytes, which were matured and co-cultured with boar spermatozoa for 2 to 6 h indicated that, at 2 to 3 h after insemination, penetrating sperm nuclei in the condensed state were not immunoreactive. At 4 to 5 h after insemination, some of the condensed sperm nuclei were immunoreactive in part or over the whole area of the nucleus, and all of the decondensing nuclei and male pronuclei were immunoreactive. At 6 h after insemination, the decondensing sperm nuclei and well-developed male pronuclei were immunoreactive. These results imply that, in pigs, remodelling of sperm nuclear protein from protamine to histone is initiated at the time of sperm penetration, before onset of decondensation and male pronuclear formation.
Effect of angiotensin II on ion transport across human Fallopian tube epithelial cells in vitro
Authors: Mahmood T , Djahanbakhch O , Burleigh DE , Puddefoot  , O'Mahony OA , Vinson GP
Published: March 2004

Angiotensin II type 1 receptors have been identified in Fallopian tube epithelia. Polarized confluent human Fallopian tube epithelial cell cultures were used under short-circuit conditions to study the actions of angiotensin II on electrogenic ion transport. The results demonstrate that angiotensin II increases baseline short-circuit current, implying a net transport of negatively charged ions from a basal to apical direction. This effect was inhibited by the selective angiotensin II type 1 receptor antagonist losartan. The effects of angiotensin II on short-circuit current were rapid in onset, brief in duration, and although less than those achieved with ATP, similar in amplitude to those described for other epithelia with angiotensin II. These findings reflect a significant retention of function for these cells in monolayer culture. Immunohistochemistry using the antibody 6313/G2, which is directed against a specific sequence in the extracellular domain of the angiotensin II type 1 receptor, confirmed that the receptor was retained in cultured cells. The results indicate that angiotensin II plays a role in regulating the composition of Fallopian tube secretions.
Regulation of ovarian function: the role of anti-Mullerian hormone
Authors: Durlinger AL , Visser JA , Themmen AP
Published: March 2004

Anti-Mullerian hormone (AMH), also known as Mullerian inhibiting substance, is a member of the transforming growth factor beta superfamily of growth and differentiation factors. In contrast to other members of the family, which exert a broad range of functions in multiple tissues, the principal function of AMH is to induce regression of the Mullerian ducts during male sex differentiation. However, the patterns of expression of AMH and its type II receptor in the postnatal ovary indicate that AMH may play an important role in ovarian folliculogenesis. This review describes several in vivo and in vitro studies showing that AMH participates in two critical selection points of follicle development: it inhibits the recruitment of primordial follicles into the pool of growing follicles and also decreases the responsiveness of growing follicles to FSH.