bluuebunny/crossref_metadata_2025 huggingface dataset:
Significance of incorporating measures of sperm production and function into rat toxicology studies
Authors: Perreault SD , Cancel AM
Published: March 2004

The rat is the preferred species for reproductive toxicity testing. The inclusion of measures of rat sperm quality, such as motility and morphology, into reproductive test protocols often increases the sensitivity of the test to detect effects, and provides the toxicologist and risk assessor with valuable information about the nature of the reproductive toxicity of the test substance. Technical advances in computer-aided sperm analysis have made it possible to evaluate motion characteristics of rat spermatozoa. This technology can provide an objective means of classifying the motion of rat spermatozoa as progressive or non-progressive, as required in test protocols. More specific tests of rat sperm function are being applied for the purpose of evaluating modes and mechanisms of toxicant action. Computer-aided sperm analysis can be used to evaluate sperm motion during cultures that support sperm capacitation and to identify hyperactivated spermatozoa. Under the same culture conditions, acrosome-specific stains can be used to identify effects of toxicants on the acrosome reaction. These approaches, in combination with in vitro fertilization in rats, can pinpoint sperm functional deficits and thereby assist the toxicologist in addressing hypotheses regarding the cellular-molecular bases of toxicant-induced male infertility.
Alterations in ovarian function of mice with reduced amounts of KIT receptor
Authors: Reynaud K , Cortvrindt R , Smitz J , Bernex F , Panthier JJ , Driancourt MA
Published: March 2004

The KIT receptor, present on oocyte and theca cells in ovarian follicles, and its ligand, KIT LIGAND, produced by granulosa cells, are encoded at the Kit gene and the Mgf gene, respectively. Both Kit and Mgf mutations affect oogenesis and folliculogenesis. In this study, the ovarian function of heterozygous mice with a mutation Kit(W-lacZ) was examined. Firstly, the amounts of KIT and KIT LIGAND proteins in the ovaries of mice at different ages were determined. Secondly, in vivo and in vitro folliculogenesis of wild type and heterozygous mice were compared. Western blotting showed that the amounts of both KIT and KIT LIGAND proteins were decreased in mutant mice. Ovarian follicle populations were counted and more type 5a follicles and fewer type 5b (preantral follicles) were present in ovaries from Kit(W-lacZ/+) ovaries. Furthermore, the relationships between oocyte size and follicle size differed between wild type and heterozygous mice. This finding may be a consequence of altered proliferation of granulosa cells or of altered oocyte growth in mutant mice. Other features of folliculogenesis, such as initiation of follicular growth, total follicle population and follicular atresia, were not affected by the mutation. Analysis of in vitro folliculogenesis did not reveal other differences between wild type and mutant mice. It is concluded that the Kit(W-lacZ) mutation affects the expression of KIT and KIT LIGAND proteins, resulting in alterations in granulosa cell proliferation and/or oocyte growth in preantral follicles.
A comparative study of sperm production in two species of Australian arid zone rodents (Pseudomys australis, Notomys alexis) with marked differences in testis size
Authors: Peirce EJ , Breed WG
Published: March 2004

The plains rat, Pseudomys australis, and the spinifex hopping mouse, Notomys alexis, show marked differences in the size of their testes and in the number of spermatozoa within the epididymides. In the present study, the dynamics of sperm production and the duration of sperm transit along the male excurrent ducts were compared between these two species. The durations of the cycle of the seminiferous epithelium, spermatogenesis and sperm transit were determined by tracking cells using autoradiography after [(3)H]thymidine incorporation. Daily sperm production was determined from counts of testicular spermatids after homogenization and further estimates of sperm transit were obtained by dividing sperm reserves within the various regions of the extratesticular ducts by the daily sperm production of the attached testis. In the plains rat, the mean duration of the cycle of the seminiferous epithelium was 11.2 days, the duration of spermatogenesis was 45 days, daily sperm production was 2.6 x 10(7) spermatozoa per gram of testis and epididymal transit of spermatozoa took approximately 9 days (caput 0.8 days; corpus 1.5 days; cauda 6.5 days). In contrast, in the hopping mouse, the mean duration of the cycle of the seminiferous epithelium was 14 days, the duration of spermatogenesis was 56 days and daily sperm production per gram of testis was < 1.0 x 10(7). Epididymal transit of spermatozoa was completed in about 4 days (caput + corpus < 1 day; cauda 3 days); however, spermatozoa may be stored for an additional 1.5-2.0 days in the vas deferens. These results indicate that, in addition to small testes, the hopping mouse shows a low efficiency of sperm production, a relatively long duration of spermatogenesis and rapid passage of spermatozoa through the epididymis, all of which contribute to low epididymal sperm counts. These data are considered in relation to interspecific differences in sperm competition.
Effect of the factor inhibiting germinal vesicle breakdown on the disruption of gap junctions and cumulus expansion of pig cumulus-oocyte complexes cultured in vitro
Authors: Isobe N , Terada T
Published: March 2004

The present study was undertaken to explore the regulatory mechanisms for meiotic resumption of pig cumulus-oocyte complexes (COCs) by assessing the nuclear status of oocytes, the degree of gap junction cell-to-cell communication and cumulus expansion after culture of various numbers of COCs in 10 microl droplets of medium for 24 h. Gap junction communication was examined by confocal laser scanning microscopy after injection of a fluorescent dye, lucifer yellow, into the oocytes. When one, three or six COCs were cultured in a 10 microl droplet, germinal vesicle breakdown was observed in > 70% of oocytes; increasing the number of COCs in a droplet further actually decreased the proportion of oocytes undergoing germinal vesicle breakdown (10 COCs: 49%; 20 COCs: 21%; 40 COCs: 13%). When six COCs were cultured in a 10 microl droplet of conditioned medium (prepared previously by culturing 20 intact and oocytectomized COCs for 24 h), the proportion of oocytes undergoing germinal vesicle breakdown was significantly reduced compared with the proportion in fresh medium. An increase in the proportion of category 1 COCs (all gap junctions within cumulus cells and between cumulus cells and oocyte are functionally maintained) was achieved by increasing the number of COCs cultured in a 10 microl droplet. The addition of conditioned medium to the fresh medium at a concentration of 50% significantly inhibited cumulus expansion. From these results, it is concluded that the factors secreted by cumulus cells regulate the disruption of gap junctions and cumulus expansion, and concurrently control the incidence of germinal vesicle breakdown in pig COCs.
Use of antibodies against LH receptor, 3beta-hydroxysteroid dehydrogenase and vimentin to characterize different types of testicular tumour in dogs
Authors: Peters MA , Teerds KJ , van der Gaag I , de Rooij DG , van Sluijs FJ
Published: March 2004

Testicular tumours in dogs are of Sertoli cell, Leydig cell or germinal origin and mixed tumours are also frequently observed. The cellular components of mixed tumours are usually identified by histological examination but sometimes this is difficult. In this study, a panel of specific antibodies was used to identify the different cell types in testicular tumours by immunohistochemistry. Leydig cells were identified using an antibody against the LH receptor and an antibody against the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD), both of which are characteristic of Leydig cells in testes. Sertoli cells were identified using an antibody against the intermediate filament vimentin. Seminoma cells did not stain with any of these antibodies. Vimentin was used only in histologically complex cases. Eighty-six tumours, diagnosed histologically as 29 Sertoli cell tumours, 25 Leydig cell tumours, 19 seminomas and 13 mixed tumours, were studied. Feminization was observed in 17 dogs. Leydig cell tumours stained positively with the antibodies against the LH receptor and 3beta-HSD, whereas seminomas and Sertoli cell tumours were negative (unstained). The antibody against vimentin stained both Sertoli and Leydig cells, and tumours arising from these cells, but not seminomas. Immunohistochemistry revealed that three tumours identified histologically as Sertoli cell tumours were actually Leydig cell tumours. In 14 dogs the histological diagnosis appeared to be incomplete, as mixed tumours instead of pure types of tumours were identified in 11 dogs, and in three dogs mixed tumours appeared to be pure types. Hence, the histological diagnosis was insufficient in approximately 20% of dogs. Furthermore, immunohistochemical analysis of testis tumours revealed that feminization occurred in dogs with Sertoli cell tumours or Leydig cell tumours and their combinations, but not in dogs with a seminoma. In conclusion, incubation with antibodies against LH receptor and 3beta-HSD proved to be a consistently reliable method for identification of Leydig cell tumours in dogs. Vimentin can be used to discriminate between Sertoli cell tumours and seminomas. Overall, this panel of antibodies can be very useful for determination of the identity of testicular tumours in which histological characterization is complicated and the pathogenesis of feminization is not clear.
Novel method for intrafollicular pressure measurements in the rat ovary: increased intrafollicular pressure after hCG stimulation
Authors: Matousek M , Carati C , Gannon B , Brannstrom M
Published: March 2004

The ovulatory process in the rat comprises a period of about 12-15 h, from the time of the preovulatory LH surge to follicular rupture and extrusion of the oocyte. Follicular rupture is most likely caused, at least in part, by decreased tensile strength at the follicular apex due to degradation of collagen fibres of the extracellular matrix. It has been debated whether changes in intrafollicular pressure occur during the ovulatory process and whether such changes facilitate rupture of the follicle. In the present study, rats were primed with equine chorionic gonadotrophin (eCG, 10 iu) followed by hCG (10 iu) 48 h later. The intrafollicular pressure in the preovulatory follicle was recorded during 1 h at distinct time phases of the ovulatory process by use of an active servo-null pressure system based on the proportionality between electrical resistance and pressure within the tip of an inserted micropipette. The basal intrafollicular pressure was 16.6 +/- 1.0 mm Hg at the preovulatory phase (48 h after eCG) and increased gradually throughout the ovulatory process to 21.4 +/- 2.4 mm Hg at 4-7 h after hCG (mid-ovulatory phase) and 23.9 +/- 1.9 mm Hg at 8-12 h after hCG (late ovulatory phase; significantly higher (P < 0.01) than the preovulatory phase). Short-term peaks of increased pressure, possibly representing contractility, were not detected in follicles of the preovulatory phase, but were seen in most follicles of the mid- and late ovulatory phases. The mean amplitude of the short-term pressure increases was 12.3 +/- 3.2 mm Hg and the increases occurred at intervals of 24.7 +/- 3.6 s. These short-term increments in intrafollicular pressure were still present after hysterectomy had been performed. The wall tension index was calculated by measuring the follicular size and estimating the thickness of the follicle wall. The index increased from 93.9 +/- 13.3 at the preovulatory phase to 207.3 +/- 47.7 (mid-ovulatory phase) and to significantly higher values at the late ovulatory phase (320.9 +/- 33.5). In conclusion, this study shows that there is an increase in intrafollicular pressure in the ovulating follicle of the rat ovary during the late stages of the ovulatory process, and that short-term increases in intrafollicular pressure occur during the late phase of the ovulatory process. These changes in pressure may be essential for follicular rupture to proceed normally.
Formation of Fallopian tubal fluid: role of a neglected epithelium
Authors: Leese HJ , Tay JI , Reischl J , Downing SJ
Published: March 2004

Fluid produced and secreted by the Fallopian tube provides the environment in which gamete transport and maturation, fertilization and early embryo development occur. This review describes the composition of oviductal fluid in terms of ions and nutrients such as glucose, lactate, pyruvate and amino acids. The function of oestrogen-specific glycoprotein is discussed. The mechanisms of fluid secretion and agents known to influence fluid production and secretion are described. Clinical implications of abnormal oviductal fluid production and secretion in hydrosalpinx and pelvic inflammatory disease are also discussed.
Isolation and partial characterization of the outer dense fibres and fibrous sheath from the sperm tail of a marsupial: the brushtail possum (Trichosurus vulpecula)
Authors: Ricci M , Breed WG
Published: March 2004

The flagellum of a mammalian spermatozoon consists of a central axoneme surrounded by two cytoskeletal structures, the outer dense fibres and the fibrous sheath, which may aid in sperm motility or stability. In this study the outer dense fibres and fibrous sheath were isolated and partially characterized in a marsupial species, the brushtail possum (Trichosurus vulpecula). Spermatozoa from the cauda epididymidis were decapitated by sonication, and the head and tail fractions were separated by centrifugation over a 20, 40 and 60% (w/v) sucrose density gradient. After confirming sperm tail purity by Nomarski microscopy, the tails were incubated in either SDS-dithiothreitol to isolate the outer dense fibres or urea-dithiothreitol to isolate the fibrous sheaths. Purified outer dense fibres and fibrous sheaths were solubilized in SDS and beta-mercaptoethanol and proteins were separated by one-dimensional PAGE. Coomassie blue staining showed that the outer dense fibres were composed of seven major proteins (molecular masses: 73, 58, 55, 54, 52, 41 and 16 kDa), and the fibrous sheath was composed of 12 major proteins (molecular masses: 106, 76, 66, 62, 55, 53, 52, 46, 40, 30, 28 and 16 kDa). A polyclonal antibody to the fibrous sheath proteins showed strong crossreactivity with those of fibrous sheath from spermatozoa of several other marsupial species, as well as those from laboratory rats. Subsequent western blotting identified the immunoreactive 76 and 62 kDa proteins from all species, thus indicating their high conservation between species. No crossreactivity of the fibrous sheath antibody to any other cytoskeletal structures, including the outer dense fibres, mid-piece fibre network or connecting laminae, or to the acrosome or underlying subacrosomal material, was evident, indicating that the fibrous sheath proteins are localized to this structure alone. Further work is in progress to determine the extent of homology of these proteins to those in eutherian mammals.
Changes in sperm quality and lipid composition during cryopreservation of boar semen
Authors: Cerolini S , Maldjian A , Pizzi F , Gliozzi TM
Published: March 2004

The effect of cryopreservation on boar sperm viability, motility, lipid content and antioxidant enzymatic activities was studied. Three classes of semen were determined according to a cluster analysis on the basis of the proportion of live and dead cells after freezing and thawing. The classes identified were: high (H, n = 4), average (A, n = 12) and low (L, n = 3) viability. The concentration of sperm cells decreased from class H to A to L. Fresh semen samples with higher viability and a higher proportion of motile cells also maintained better quality after the freezing and thawing procedure. Sperm viability and motility in both fresh and thawed samples were similar in classes H and A, while significantly lower values were measured in class L. The relative decrease in sperm viability and motility after cryopreservation increased from class H to A to L. The lipid content of spermatozoa (micrograms per 10(9) cells) increased significantly after freezing and thawing in classes H and A but not in class L. This result indicated that active sperm lipid metabolism might be responsible for the increase in lipid content. Phospholipid and triacylglycerol contents increased whereas free cholesterol content decreased after thawing. The fatty acid composition of fresh spermatozoa was similar in all three classes. The proportion of polyunsaturated fatty acids decreased significantly after freezing and thawing, indicating contamination from the diluent or peroxidation. After freezing and thawing, superoxide dismutase activity in spermatozoa was significantly higher in class L than in classes H and A, which did not differ from each other.
Influence of genetic dissimilarity of mother and fetus on progesterone concentrations in pregnant mice and adaptive features of offspring
Authors: Gerlinskaya LA , Evsikov VI
Published: March 2004

Concentrations of progesterone in blood plasma and tissue were studied in pregnant mice of strains BALB/cLac and C57BL/6J. Both intra- and interstrain mating and embryo transplantations were used as models of homo- and heterotopic pregnancy. On day 4 of heterotopic pregnancy, plasma progesterone concentrations of females of both strains were higher than those in females of both strains undergoing homotopic pregnancy. In addition, tissue progesterone content of hybrid embryos was higher than that of purebred embryos. Adrenocortical responses to social conflict as indicators of stress resistance were studied in progeny aged 2-3 months. There was a minimal increase in plasma glucocorticoid concentrations in heterotopic transplantants after 15 min pair-matching tests and 30 min crowding compared with those of other progeny, including purebred male mice of BALB/cLac and C57BL/6J strains, homotransplantants and reciprocal hybrids. Thus, genetic dissimilarity of mother and fetuses plays an important role in progesterone provision during pregnancy and also modifies development of progeny.
Ultrasonographic imaging of the reproductive organs of the female bottlenose dolphin, Tursiops truncatus aduncas
Authors: Brook FM
Published: March 2004

Routine ultrasonographic examination of the reproductive tract was performed for periods of up to 10 years in ten female bottlenose dolphins (Tursiops truncatus aduncas) in Hong Kong. The ovaries could be reliably and repeatedly identified, lying close to the body surface, in the angle formed by the rectus abdominus and hypaxialis lumborum muscles, and were most easily located by scanning in the transverse plane from the proximal end of the genital slit towards the head. The ovaries are ovoid, with a relatively hypoechoic cortex around a central echogenic mesovarium. The echogenicity of the ovarian parenchyma appeared to increase with increasing age. This may be the result of age-related changes, such as increased fat deposition or fibrosis, or of ovarian 'scars' from multiple ovulations. Small antral follicles, developing follicles and corpora lutea can be identified within the ovarian cortex. Owing to its shape and lack of a definitive border, plus the close relationship to the intestines, the contents of which may obstruct the ultrasound beam, the non-pregnant uterus was not so easily visualized. The endometrium was poorly differentiated and difficult to see. To date, examination of the uterus using ultrasonography has provided little information about endometrial changes during the ovarian cycle in this group of dolphins. Real-time diagnostic ultrasonography provides a means to image the morphology of the reproductive organs in live female dolphins directly and provides a valuable means of assessing reproductive events in this species.
Synthesis and glycosylation of CD52, the major 'maturation-associated' antigen of rat spermatozoa, in the cauda epididymidis
Authors: Derr P , Yeung CH , Cooper TG , Kirchhoff C
Published: March 2004

A western and lectin blot analysis was performed of the major 'maturation-associated' antigen of rat spermatozoa, which is the rat counterpart of human CD52. In the absence of a suitable antibody, direct study of this approximately 26 kDa antigen, named previously SMemG, had been difficult. In the present study, these problems were overcome by raising a polyclonal antibody against a chemosynthetic peptide predicted from the cDNA sequence of the antigen. The antibody bound to a glycoprotein of rat cauda epididymidal tissue and spermatozoa, this glycoprotein was cleaved by phosphatidylinositol-specific phospholipase C and, after deglycosylation, was reduced to approximately 6 kDa. Northern blot analysis confirmed that the CD52 mRNA was transcribed only post-testicularly, and antibody binding to testicular and sperm proteins of different molecular masses was shown to be nonspecific. Flow cytometry also indicated that the antigen was inserted into the sperm membrane during epididymal transit. Moreover, despite the presence of CD52 mRNA in all parts of the rat epididymis, only the 'long' mRNA molecules of the cauda region were efficiently translated and the antigen glycosylated, indicating that expression of rat CD52 is regulated on a post-transcriptional level. Lectin binding and deglycosylation studies supported the contention that there is extensive mucin-type O-glycosylation of rat CD52. In rats, there was no indication of complex N-linked carbohydrates similar to those described for human CD52.
Differences in follicular function of 3-month-old calves and mature cows
Authors: Driancourt MA , Reynaud K , Smitz J
Published: March 2004

After in vitro maturation, fertilization and development, the percentage of fertilized eggs developing to the blastocyst stage is usually lower in calves compared with cows. It is unknown whether this low ability to develop in vitro is inherent to calf oocytes or is caused by altered follicular maturation. The latter possibility was explored in the present study using two markers of follicle function: in vitro steroidogenesis by intact follicles and aromatase activity of follicular walls. Calf follicles > 9 mm in diameter had a low ability to produce oestradiol (ten times reduction compared with cows) despite a testosterone output by theca cells which was similar to that observed in cows. This finding is in agreement with the low aromatase activity of granulosa cells of calf follicles measured by tritiated water release assay. Qualitative and quantitative differences between calf and cow follicular fluids were assessed using western blotting (inhibin and activin, heat shock protein 90, Mullerian inhibiting substance) and assays (inhibin and activin) to determine whether this defective aromatase could be produced by alterations in the amounts of follicular proteins modulating aromatase (inhibin and activin, heat shock protein 90, Mullerian inhibiting substance). Western blotting of follicular fluid proteins demonstrated three main bands (59, 57 and < 30 kDa) and one minor band (34 kDa) with the anti-alpha inhibin antibody, whereas a single 18 kDa band was detected when an anti-beta inhibin antibody was used. Calf follicular fluid contained similar amounts of all main inhibin forms (alpha and beta) but a 34 kDa alpha inhibin form was missing. The amounts of dimeric inhibin were similar between cows and calves but small follicles from calves contained more activin. Single bands at 70 kDa (Mullerian inhibiting substance) and 90 kDa (heat shock protein 90) were detected by western blotting. Mullerian inhibiting substance was missing from calf follicular fluid and heat shock protein 90 was present in smaller amounts in calf versus cow follicular fluid. None of the above differences could explain the defective aromatase of calf follicles. Two-dimensional separation of the [35S]-labelled proteins secreted by follicular walls originating from calf or cow follicles matched for size and follicle health was performed and 151 spots were observed on the master gel, which summarized all the spots present at least once. Fifteen spots were present in calves and not in cows. Quantitative differences were also detected with three spots containing more proteins in cows than in calves. Whether some of these proteins can alter maturation of follicles or oocytes requires further investigation.
Inhibition of basal and stimulated progesterone synthesis by dichlorodiphenyldichloroethylene and methoxychlor in a stable pig granulosa cell line
Authors: Crellin NK , Kang HG , Swan CL , Chedrese PJ
Published: March 2004

The effects of the insecticide dichlorodiphenyldichloroethylene (DDE) and methoxychlor in a stable pig granulosa cell line, JC-410, were investigated. The studies of DDE and methoxychlor were conducted in combination with studies of cholera toxin, the protein kinase A activator that stimulates cAMP and progesterone synthesis and gene expression of P450 cholesterol side chain cleavage (P450scc), which converts cholesterol to pregnenolone. Administration of DDE at 3000 and 10 000 ng ml (-1) was found to decrease progesterone synthesis 0.49- and 0.25-fold, respectively, and to block the stimulatory effect of 100 ng cholera toxin ml (-1), after 24 h incubation. At 1-100 ng ml (-1), methoxychlor did not affect progesterone synthesis after 48 h incubation. However, 1000 ng methoxychlor ml (-1) decreased progesterone synthesis 0.32-fold, and both 100 and 1000 ng methoxychlor ml (-1) blocked the stimulatory effect of cholera toxin. At 3000 and 10 000 ng ml(-1), DDE decreased cAMP synthesis 0.66-and 0.36-fold, respectively. At 300, 3000 and 10 000 ng ml (-1), DDE also decreased cholera toxin-stimulated cAMP synthesis 0.84-, 0.68-, and 0.52-fold, respectively. Administration of 1-100 ng methoxychlor ml (-1) did not affect basal or cholera toxin-stimulated cAMP synthesis. Cholera toxin increased P450scc mRNA 1.4-fold after 24 h incubation, while 3000 and 10 000 ng DDE ml (-1) led to 0.39- and 0.18-fold reductions, respectively. The stimulatory effect of cholera toxin on P450scc mRNA was blocked by 3000 and 10 000 ng DDE ml(-1). Cholera toxin increased P450scc mRNA 3.48-fold after 48 h incubation, while 100 and 1000 ng methoxychlor ml (-1) increased P450scc mRNA 1.79- and 3.0-fold, respectively, and further increased the stimulatory effect of cholera toxin 6.47- and 5.44-fold, respectively. The results of the present study indicate that DDE inhibits granulosa cell steroidogenesis by affecting cAMP production and P450scc gene expression. However, methoxychlor appears to inhibit steroidogenesis by a mechanism occurring before the conversion of cholesterol into pregnenolone.
Corticotrophin-releasing hormone and human parturition
Authors: McLean M , Smith R
Published: March 2004

Corticotrophin-releasing hormone (CRH), the hypothalamic peptide that controls function of the pituitary-adrenal axis in response to stress, is expressed in abundance in the human placenta and is present in high concentrations in maternal and fetal plasma during late pregnancy. A number of lines of evidence now imply a role for this hormone in the control of parturition and fetal maturation in humans. It has been proposed that CRH, through interactions with oestrogen, adrenal steroids, prostaglandins and oxytocin, establishes positive-feedback loops that initiate parturition and labour. Excessive production of placental CRH has also been linked to preterm labour. However, there are a number of significant gaps in our knowledge of the function of this peptide in pregnancy. This review examines current evidence regarding the putative role of CRH in human parturition.
Fetomaternal interactions and influences during equine pregnancy
Authors: Allen WR
Published: March 2004

The equine embryo takes 6 days to traverse the oviduct and, when it finally enters the uterus, it remains spherical in shape and moves continually throughout the uterine lumen until day 17 after ovulation to deliver its maternal recognition of pregnancy signal to the entire endometrium. Between day 25 and day 35 after ovulation, the trophoblast cells of a discrete annulate portion of the chorion multiply rapidly and acquire an invasive phenotype and, between day 36 and day 38, migrate deeply into the maternal endometrium to form the equine-unique endometrial protuberances known as endometrial cups. These cups secrete large quantities of a gonadotrophic hormone (eCG) into the maternal circulation which, in conjunction with pituitary FSH, stimulates the development of accessory luteal structures in the maternal ovaries to supplement the supply of progesterone to maintain the pregnancy until the placenta can assume this role at about day 100. The non-invasive allantochorion extends slowly to fill the uterus by days 80-85 and its microcotyledonary architecture, which provides both haemotrophic and histotrophic nutrition for the growing fetus, is not fully established until days 120-140. The fetoplacental unit synthesizes large quantities of steroid hormones during the second half of pregnancy, using fetal C-19 precursors secreted by the enlarged fetal gonads for the production of oestrogens and maternal C-21 precursors for the synthesis of progesterone and large quantities of 5alpha-reduced progestagens. Near term, additional pregnenelone is secreted by the fetal adrenal glands so that the mare exhibits the unusual phenomenon of foaling while maternal serum progestagen concentrations are increasing and oestrogen concentrations are decreasing.
Chemosensation and genetic individuality
Authors: Singh PB
Published: March 2004

Numerous studies have shown that there are measurable behavioural consequences that can result from the olfactory recognition of alleles borne at the major histocompatibility complex (MHC). These consequences include simple individual recognition, disassortative mate preference, discrimination of kin from non-kin and whether a pregnancy is carried to term. Such a system, which can influence the reproductive behaviour of a species, will have profound effects on its genetic constitution and survival. The likely mechanism responsible for the production of MHC-related odours involves soluble MHC molecules that carry allele-specific odoriferous molecules from the blood via the kidneys into the urine, from where they are released into the environment. The ability of soluble MHC molecules to signal genetic individuality in this way may have evolved before the appearance of an acquired immune system in our immediate ancestors, the protochordates.
Inhibition of neutrophil chemotaxis by pig seminal plasma in vitro: a potential method for modulating post-breeding inflammation in sows
Authors: Rozeboom KJ , Rocha-Chavez G , Troedsson MH
Published: March 2004

The aim of this study was to determine the regulatory role of pig seminal plasma in post-breeding uterine inflammation. Polymorphonuclear neutrophil (PMN) chemotaxis of lipopolysaccharide (LPS)-activated blood plasma or heat-inactivated blood plasma plus LPS containing increasing concentrations of seminal plasma was assessed in chemotactic chambers. Seminal plasma was diluted serially with McCoy's medium to concentrations of 50.0, 25.0, 12.5, 6.2 or 3.1% (v/v) and added to normal or heat-inactivated pig blood plasma that was activated with LPS before or after incubation in a 37 degrees C waterbath for 30 min. Chemotaxis was determined using blood-derived PMNs and was expressed as a percentage of the positive control of LPS-activated blood plasma. A linear dose-dependent suppression of chemotaxis by seminal plasma was observed for blood plasma activated before or after addition of seminal plasma. Compared with the positive control, concentrations of seminal plasma < 6.2% failed to suppress PMN chemotaxis (P < 0.05). A dose-dependent suppressive effect of seminal plasma on heat stable chemotactic components of pig blood plasma was also observed (P < 0.05). A marked suppression was observed at concentrations of seminal plasma > 12.5% of the sample volume (P < 0.05). These results indicate that seminal plasma suppresses chemotactic blood plasma components regardless of formation sequence (pre- or post-activation) or source (normal or heat-inactivated blood plasma). These results indicate that seminal plasma may be necessary in diluted boar semen used for artificial insemination to regulate post-breeding inflammation in sows.
Immunolocalization of steroidogenic enzymes in the corpus luteum and placenta of the Japanese black bear, Ursus thibetanus japonicus, during pregnancy
Authors: Tsubota T , Taki S , Nakayama K , Mason JI , Kominami S , Harada N , Kita I
Published: March 2004

The Japanese black bear, Ursus thibetanus japonicus, is a seasonal breeder and shows delayed implantation for several months during pregnancy. The objective of this study was to clarify the steroidogenic capability of the corpus luteum and placenta during pregnancy, including both delayed implantation and fetal development, by immunolocalization of steroidogenic enzymes in these organs of the Japanese black bear. Ovaries and placentae from 15 wild Japanese black bears, which had been killed legally by hunters and were thought to be pregnant, were used in an immunocytochemical study to localize the cholesterol side chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17alpha-hydroxylase cytochrome P450 (P450c17) and aromatase cytochrome P450 (P450arom) by the avidin-biotin-peroxidase complex method using polyclonal antisera raised in mammals against P450scc, 3betaHSD, P450c17 and P450arom. P450scc and 3betaHSD were localized in all luteal cells throughout pregnancy. P450c17 was present in a few luteal cells, especially in the outer area of the corpus luteum throughout pregnancy, but the number of positively immunostained cells decreased during the post-implantation period. Cells positively immunostained for P450c17 were significantly smaller than negatively immunostained cells (P < 0.01). P450arom was present sporadically in a few luteal cells throughout pregnancy, but the number of positively immunostained cells decreased during the post-implantation period. The size of cells positively immunostained for P450arom was not significantly different from that of negatively immunostained cells. The whole placenta was negatively immunostained for P450scc, 3betaHSD and P450c17, but P450arom was present in the syncytiotrophoblasts and endothelial cells of maternal blood vessels. These results indicate that, in the Japanese black bear, corpora lutea are a source of progesterone which may play an important role in the maintenance of delayed implantation and fetal development during pregnancy. Corpora lutea have a minimum capability to synthesize androgen in small luteal cells and oestrogen in normal-sized luteal cells during pregnancy, and placentae have the ability to synthesize oestrogen during late pregnancy.
Reproductive seasonality in domestic sows kept outdoors without boars
Authors: Bassett JM , Bray CJ , Sharpe CE
Published: March 2004

Plasma progesterone, LH and prolactin concentrations were measured twice a week in mature sows kept outdoors without boars in two experiments to examine whether perception of daylength change underlies seasonal infertility in domestic pigs. In Expt 1, melatonin implants inserted on 12 April or 22 May to block perception of the increasing daylength did not affect the oestrous cycle significantly, since only two untreated control sows became seasonally anoestrous. In Expt 2, all control sows became anoestrous for 131 +/- 42.5 days (n = 11). Melatonin implants inserted at the spring equinox (n = 9) prevented seasonal anoestrus (P < 0.001), but timed daily oral melatonin administration was less effective (P < 0.05): 5 of 11 sows became anoestrous for 132 +/- 45.6 days. In both experiments, there were significant low-amplitude seasonal rhythms in mean plasma prolactin and LH concentrations. Prolactin reached maximum concentrations 2-4 weeks before the summer solstice, whereas LH reached a nadir 4-6 weeks after the summer solstice. Neither anoestrus nor melatonin administration altered these patterns consistently. Endogenous plasma melatonin was higher (P < 0.001) during darkness (12.5 ng l(-1)) than during daytime (8.9 ng l(-1)) in untreated sows, but only clearly so during spring and summer. Melatonin implants increased mean daily plasma melatonin to 146 ng l(-1), whereas melatonin fed at 15:00 h increased values to 40-60 ng l(-1) 2-4 h after administration, but daytime concentrations were unchanged. Melatonin administration, despite decreasing seasonal anoestrus, did not prevent the seasonal increase in plasma prolactin and had no significant effect on plasma LH; therefore, its role in regulating seasonal changes in the reproduction of domestic sows remains uncertain.
Regulation of lipid peroxidation by nitric oxide and PGF2alpha during luteal regression in rats
Authors: Motta AB , Estevez A , Franchi A , Perez-Martinez S , Farina M , Ribeiro ML , Lasserre A , Gimeno MF
Published: March 2004

Corpus luteum regression is related to an increased generation of reactive oxygen species. Although several studies indicate that PGF(2alpha) is involved in regression of the corpus luteum in mammalian species through an increase in reactive oxygen species, the exact mechanism remains unknown. In the present study, the relationship between nitric oxide and PGF(2alpha) in regulation of lipid peroxidation was studied. Ovarian tissue from pseudopregnant rats at mid- (day 5) or late phase or at the time of regression (day 9 of pseudopregnancy) of corpus luteum development was used. Thiobarbituric acid reactants, used as a lipid peroxidation index, were higher on day 9 of pseudopregnancy than on day 5. In contrast, glutathione content (an antioxidant metabolite) was lower on day 9 than on day 5 of pseudopregnancy. These results indicate that there was an enhanced oxidative status in ovarian tissue during luteolysis. Administration of N(omega)-nitro-L-arginine methyl ester (L-NAME: 600 micromol l(-1)), a competitive nitric oxide synthase (NOS) inhibitor, led to a decrease in basal thiobarbituric acid reactant content in ovarian tissue from rats on day 9 of pseudopregnancy only, indicating that during regression of the corpus luteum, NO could act as intermediary in ovarian lipid peroxidation. Administration of a luteolytic dose (3 microg kg(-1) body weight i.p.) of a synthetic PGF(2alpha) increased thiobarbituric acid reactant content in ovaries from rats on day 9 of pseudopregnancy. As this effect was reversed partially by L-NAME, it is proposed that during regression of corpora lutea, PGF(2alpha) and NO are involved in regulation of lipid peroxidation. As this effect was only reversed partially, it is possible that there is another mechanism involving PGF(2alpha) (but not the NO-NOS pathway) in regulation of ovarian lipid peroxidation. Furthermore, the administration of PGF(2alpha) enhanced ovarian NOS activity, whereas cyclooxygenase inhibition (by indomethacin treatment in vivo) reduced it. As western blotting of ovarian homogenates obtained from PGF(2alpha)-injected rats increased inducible NOS (iNOS) content, it is concluded that PGF(2alpha) enhances both activity and synthesis of NO in rat ovarian tissues during luteolysis. Taken together, these results indicate that in ovaries with regressing corpora lutea, both NO and PGF(2alpha) are involved in part in regulation of lipid peroxidation.
Effect of transient early hyperthyroidism on onset of puberty in Suffolk ram lambs
Authors: Fallah-Rad AH , Connor ML , Del Vecchio RP
Published: March 2004

The thyroid function and sexual development of eight 6-week-old Suffolk ram lambs were studied. The lambs were divided into either control or treatment groups and housed indoors. From 6 to 12 weeks of age, four lambs in the treatment group received 15 mg kg(-1) body weight per day of 6-propyl 2-thiouracil orally to suppress normal thyroid function. During the same period, thyroxine and tri-iodothyronine were injected s.c. at the rate of 8 and 16 microg kg(-1) body weight per day, respectively, to induce a hyperthyroid state. Four control lambs received sham injection and oral excipient. Concentrations of thyroxine, tri-iodothyronine, FSH, testosterone and insulin-like growth factor I were determined in blood collected by indwelling jugular catheters once a week, every 20 min from 09:00 to 15:20 h. Scrotal circumference was recorded each week. Semen collection was attempted by electro-ejaculation between weeks 17 and 36. Lambs were castrated at week 36 and testicular histology was examined. During the treatment period only, the concentration of thyroid hormones was higher in treated lambs than in controls (P < 0.05). From week 6 to week 9 only, concentrations of FSH in treated lambs were lower than in controls (P < 0.05). Insulin-like growth factor I concentrations were lower in treated lambs than in controls from week 10 to week 13 (P < 0.05). Frequency of testosterone pulses was higher (P < 0.01) in the treated lambs but concentrations were similar in the control and treated lambs throughout the experiment. Scrotal circumference was greater in treated lambs from week 26 to week 36 (P < 0.05). Treated lambs produced viable spermatozoa earlier than did control lambs. At week 36, sperm concentration in treated lambs was higher than in controls (P < 0.01) but semen volumes were similar (P > 0.1). Diameter of the seminiferous tubules in treated lambs was larger than in controls (P < 0.05). In conclusion, transient neonatal hyperthyroidism decreased FSH and insulin-like growth factor I concentrations temporarily, increased testosterone pulses and sperm production and advanced puberty in Suffolk ram lambs.
Aneuploidy in human spermatozoa: FISH analysis in men with constitutional chromosomal abnormalities, and in infertile men
Authors: Shi Q , Martin RH
Published: March 2004

Reproductive difficulties are associated intimately with cytogenetic abnormalities. This article reviews multicolour fluorescence in situ hybridization studies on spermatozoa from men with constitutional chromosomal abnormalities and the consequences for spermatozoa, and on chromosomal abnormalities in the spermatozoa of infertile men who have normal somatic karyotypes. In 47,XYY men, the frequencies of 24,XY and 24,YY spermatozoa appear to be < or = 1%. Klinefelter (47,XXY) and mosaic Klinefelter patients had sperm aneuploidy frequencies of 2-25% and 1.5-7.0%, respectively. Robertsonian translocation carriers had 3-27% spermatozoa unbalanced for the chromosomes involved in the translocation, with a possible modest interchromosomal effect, but none of the increased frequencies of chromosomal disomy approached 1%. The frequency of chromosomally unbalanced spermatozoa in reciprocal translocations averages 50%, is strongly dependent on the chromosomes involved in the individual translocation, and may be slightly increased as a result of a small interchromosomal effect. Infertile men with a normal karyotype and low sperm concentration or certain types of morphologically abnormal spermatozoa have a significantly increased risk of producing aneuploid spermatozoa, particularly for the sex chromosomes. An increased risk of sperm aneuploidy was not observed in infertile men with poor sperm motility or in those with a normal karyotype and normal semen parameters.
Roles of activin and its signal transduction mechanisms in reproductive tissues
Authors: Ethier JF , Findlay JK
Published: March 2004

Activins were identified initially as gonadal proteins having a stimulating effect on FSH production by the pituitary gland. Strong evidence has accumulated that activins are important regulating factors for many reproductive processes. Activin may have paracrine or autocrine roles rather than solely an endocrine action on FSH secretion. Activins together with their signalling molecules must be shown to be produced locally in a particular tissue to provide support for their paracrine or autocrine action in that tissue. The discovery of the activin receptors, the intracellular signalling mediators (Smads) and some transcription co-factors involved in activin responses has helped to unravel the activin-transforming growth factor beta signalling mechanism. However, few reports have clearly demonstrated the presence of all of the activin signalling molecules in reproductive tissues, despite the important roles of activin in these tissues. Several activin receptor types and Smad molecules have been identified, indicating either a redundancy in signalling molecules or different signalling pathways. At present, it is not clear which particular subset of these signalling molecules is important in reproductive processes. The aim of this review is to collate the information available on activin actions, as well as on the signalling molecules, to understand how activins may transduce their paracrine or autocrine signals in reproductive tissues.
Possible role of kallikrein in proteolysis of insulin-like growth factor binding proteins during the oestrous cycle and early pregnancy in pigs
Authors: Geisert RD , Chamberlain CS , Vonnahme KA , Malayer  , Spicer LJ
Published: March 2004

During early pregnancy, pig conceptuses initiate the synthesis of oestrogens and on day 12 their trophoblastic membranes undergo a rapid expansion throughout the uterine horns. The insulin-like growth factor (IGF) system may be involved with conceptus development and steroidogenesis in pigs. Changes in uterine luminal IGF, insulin-like growth factor binding proteins (IGFBPs) and enzymatic activity for cleavage of IGFBPs during the oestrous cycle and early pregnancy were investigated. Uterine luminal content of IGF-I and IGF-II in uterine flushings from pigs on day 12 of pregnancy were two and three times greater, respectively, compared with uterine flushings collected from gilts during the oestrous cycle. Both IGF-I and -II content decreased on day 15 of gestation but content of IGF-II in uterine flushings remained three times greater than that of cyclic gilts. IGFBP-2 and -3 were the predominant binding proteins present in uterine flushings during days 0-10 of the oestrous cycle or day 10 of pregnancy. No IGFBPs were detected in the uterine flushings of either cyclic or pregnant pigs after day 10 by ligand blotting. Incubation of [125I]-labelled IGFBPs with various protease inhibitors indicated that cleavage of [125I]-labelled IGFBP-2 and -3 in uterine flushings involved serine proteases such as tissue kallikrein and matrix metalloproteinases. The results of the present study indicate that an increase in tissue kallikrein activity on day 12 of the oestrous cycle and pregnancy in pigs can directly, or indirectly through activation of matrix metalloproteinases, cleave IGFBP-2 and -3, thus allowing uterine release of IGF-I and -II in the uterine lumen to stimulate conceptus development.
Full-term development of enucleated mouse oocytes fused with embryonic stem cells from different cell lines
Authors: Amano T , Kato Y , Tsunoda Y
Published: March 2004

The developmental potential of enucleated mouse oocytes receiving embryonic stem cells from ten lines with either the same or different genetic backgrounds using the cell fusion method was examined in vitro and in vivo. The development of nuclear-transferred oocytes into blastocysts was high (34-88%). However, there was no clear correlation between development into blastocysts after nuclear transfer and the chimaera formation rate of embryonic stem cells. The development into live young was low (1-3%) in all cell lines and 14 of 19 young died shortly after birth. Most of the live young had morphological abnormalities. Of the five remaining mice, two died at days 23 and 30 after birth, but the other three mice are still active at days 359 (mouse 1) and 338 (mice 4 and 5) after birth, with normal fertility. However, the reasons for the abnormalities and postnatal death of embryonic stem cell-derived mice are unknown.