bluuebunny/crossref_metadata_2025 huggingface dataset:
Potential local regulatory functions of inhibins, activins and follistatin in the ovary
Authors: Knight PG , Glister C
Published: March 2004

The changing pattern of granulosa cell expression of inhibin/activin subunits and follistatin during follicle development and their differential regulation by extrinsic and intraovarian factors supports evidence from functional studies, mostly in vitro, that these proteins have important roles in folliculogenesis, oocyte maturation and corpus luteum function. Gonadal inhibins function as negative feedback hormones to regulate the synthesis and secretion of pituitary FSH, a key determinant of follicle development, but there is little supportive evidence for a peripheral endocrine role for ovary-derived activins or follistatin in this regard. However, activins and follistatin are expressed in numerous other tissues, including anterior pituitary, and they are firmly implicated as local intrapituitary regulators of FSH secretion. Intraovarian actions of granulosa cell-derived activins include the promotion of granulosa cell proliferation and upregulation of FSH receptors, P450arom, oestrogen synthesis, granulosa cell LH receptors and enhancement of oocyte maturation. Through its activin-binding role, follistatin can reverse each of these activin-induced responses. In addition to their endocrine feedback role, granulosa-derived inhibins can sensitize theca cells to LH, thereby enhancing the production of androgens, an essential requirement for follicular oestrogen synthesis. Activins can oppose this effect and suppress thecal androgen production. Granulosa cells overproduce inhibin a subunit precursor relative to betaA/betaB subunit precursors and evidence indicates that different parts of the inhibin a subunit precursor have intrinsic biological activities distinct from inhibin alphabetaA/B dimer, and serve as additional local modulators of follicle and corpus luteum function.
An X-Y paint set and sperm FISH protocol that can be used for validation of cattle sperm separation procedures
Authors: Rens W , Yang F , Welch G , Revell S , O'Brien PC , Solanky N , Johnson LA , Ferguson Smith MA
Published: March 2004

X and Y chromosome paints were developed from sorted yak chromosomes for sexing cattle spermatozoa. Clear hybridization signals were obtained for every spermatozoon using a modified sperm decondensation protocol and fluorescence in situ hybridization (FISH). The procedure was evaluated using the established Beltsville sperm sexing technology, which separates spermatozoa by flow cytometry into X- and Y-bearing fractions. Close agreement was found between the assessment of sperm separation by flow cytometry and by FISH with the X-Y paint set. The FISH method is a simple, reliable and robust procedure for assessing the effectiveness of separation of X and Y spermatozoa.
Localization of primordial germ cells or their precursors in stage X blastoderm of chickens and their ability to differentiate into functional gametes in opposite-sex recipient gonads
Authors: Naito M , Sano A , Matsubara Y , Harumi T , Tagami T , Sakurai M , Kuwana T
Published: March 2004

This study was performed to determine the distribution of primordial germ cells and their precursors in stage X blastoderm of chickens. The blastoderm (Barred Plymouth Rock chickens) isolated from the yolk was separated into three portions: the central disc, the marginal zone and the area opaca. The dissociated blastodermal cells derived from the central disc, marginal zone and area opaca were transferred into a recipient blastoderm (White Leghorn chicken) from which a cell cluster was removed from the centre of the central disc. The manipulated embryos were cultured in host eggshells until hatching. The chicks were raised until sexual maturity and test mated with Barred Plymouth Rock chickens to assess the donor cell contribution to the recipient germline. Germline chimaeric chickens were produced efficiently (46.7%, 7/15) when the blastodermal cells derived from the central disc were transferred into recipient embryos of the same sex, whereas no germline chimaeric chickens were produced when the blastodermal cells derived from the marginal zone or area opaca were transferred into recipient embryos of the same sex (0/12). Germline chimaeric chickens were also produced by transfer of blastodermal cells derived from the central disc (6.7%, 1/15), marginal zone (10.0%, 1/10) or area opaca (11.1%, 1/9) into recipient embryos of the opposite sex. It is concluded that primordial germ cells are induced during or shortly after stage X and that the cells derived from the central disc have the highest potential to give rise to germ cells. Cells derived from the marginal zone and area opaca can also give rise to germ cells, although the frequency is low.
Secretion of matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases into follicular fluid during follicle development in equine ovaries
Authors: Riley SC , Gibson AH , Leask R , Mauchline DJ , Pedersen HG , Watson ED
Published: March 2004

Extensive tissue remodelling is required in equine ovaries for follicle growth and development and also migration of the follicle to the ovulatory fossa, where ovulation occurs. The mechanisms for these processes are largely unexplored. Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) are important for control of breakdown of extracellular matrix during tissue remodelling. The aims of this study were to determine the pattern and sites of secretion of the gelatinases MMP-2 and -9 and TIMPs into follicular fluid during follicle development in mare ovaries. The predominant gelatinase detected in follicular fluid was MMP-2, which was present in similar amounts throughout follicular development, as demonstrated by zymography. MMP-9 was also present in follicular fluid and secretion increased significantly (P < 0.05) with development of follicles from < 10 mm to 11-20 mm in diameter. Follicular fluid also contained TIMP-1, TIMP-2, unglycosylated and glycosylated TIMP-3, and TIMP-4, as shown by reverse zymography. The abundance of TIMPs remained largely unchanged during follicle development. MMP-2 and -9 were localized by immunohistochemistry to stromal cells and granulosa and theca cells. TIMP-1, -2, -3 and -4 were present in granulosa and theca cells of the follicle and in stromal cells and also associated with extracellular matrix of the ovarian stromal tissue. The MMPs and TIMPs are likely to be involved in the regulation of the breakdown of extracellular matrix during tissue remodelling for follicle development and migration to the ovulation fossa in mares.
Relationship of Fas ligand expression and atresia during bovine follicle development
Authors: Porter DA , Harman RM , Cowan RG , Quirk SM
Published: March 2004

The Fas antigen (Fas) is a cell surface receptor that may be involved in the initiation and progression of follicle cell apoptosis during atresia. Fas initiates apoptosis in sensitive cells after binding Fas ligand (FasL). Other experiments have shown that expression of Fas mRNA and responsiveness to Fas-mediated apoptosis vary in bovine granulosa and theca cells during follicle development. In the present study, FasL mRNA content was measured and Fas and FasL protein expression was examined in bovine granulosa and theca cells of healthy dominant follicles and the two largest atretic subordinate follicles on day 5 of the oestrous cycle (day 0 = oestrus), and of dominant follicles from the first wave of follicle development after they had become atretic and showed no growth for 4 days. FasL mRNA content was higher in granulosa cells from atretic compared with healthy follicles. FasL mRNA content was also higher in theca cells from atretic subordinate compared with healthy dominant follicles on day 5, but did not differ between theca cells from healthy and atretic dominant follicles. Immunohistochemical staining for FasL was more intense in theca compared with granulosa cells and in atretic compared with healthy follicles. Immunohistochemical staining for Fas was more intense in granulosa compared with theca cells and in atretic subordinate compared with healthy dominant follicles on day 5. Immune cells, known to express Fas and FasL, were localized in the theca, but not the granulosa, cell layer of all follicles. Higher concentrations of Fas and FasL in cells from atretic follicles, together with the previous demonstration of increased responsiveness of granulosa cells from subordinate follicles to FasL-induced apoptosis, support a potential role for FasL-mediated apoptosis during ovarian follicle atresia.
Expression of heparanase mRNA in bovine placenta during gestation
Authors: Kizaki K , Nakano H , Takahashi T , Imai K , Hashizume K
Published: March 2004

This study reports the identification and sequence of a partial cDNA for bovine heparanase and the expression of its mRNA in the placenta during gestation. The 364 amino acid residues deduced from the 1092 bp cDNA fragment share 81.9% and 80.5% identity with amino acid sequences of human and rat heparanase, respectively. Northern blot hybridization showed that two mRNAs (2.0 and 3.5 kb) are strongly expressed in placenta, and weakly expressed in the kidney, lung, spleen and non-pregnant uterus. In the placenta, these transcripts were detected in the cotyledon at all stages of gestation examined, and in the intercotyledonary fetal membrane and caruncle on day 60, day 120 and day 260. Quantitative real-time RT-PCR analysis showed very low expression of heparanase mRNA in the conceptus before implantation (day 17), but high expression in the cotyledon-containing fetal membrane (days 27-34) after implantation. Furthermore, heparanase mRNA was detected in the cotyledon, intercotyledonary fetal membrane and caruncle after days 60-64 of gestation. However, no significant expression of heparanase mRNA was observed in intercaruncular endometrium at all stages of gestation examined. These results demonstrate that heparanase mRNA is expressed in the placentome, indicating that heparanase may play a role in implantation, and in placental development and function.
Association of endometriosis in horses with differentiation of periglandular myofibroblasts and changes of extracellular matrix proteins
Authors: Walter I , Handler J , Reifinger M , Aurich C
Published: March 2004

Periglandular fibrosis and cystic dilation of uterine glands are associated with equine endometriosis. The presence of extracellular matrix proteins (collagen type I, III and IV, laminin and fibronectin) in healthy and endometriotic specimens was demonstrated by immunohistochemistry. The distribution of collagen I, but not collagen III, was dependent on the stage of the oestrous cycle. The arrangement of collagen I and collagen III in endometriotic specimens was similar to that in normal endometrium. In periglandular fibrosis, collagen IV, laminin and fibronectin deposition outside the basement membrane was observed. In these regions, stromal cells were characterized immunohistochemically as myofibroblasts because of their expression of a-smooth muscle actin, and occasionally tropomyosin and desmin. Periglandular differentiation of contractile cells could be interpreted as a reaction to support the extrusion of secretions in cystic dilated glands. Moreover, the changes of extracellular matrix proteins are characteristic for neoplastic lesions, although further development of endometriosis to benign or malignant tumours is not known in horses. Knowledge of the factors responsible for these fibroblastic modulations may be the key to explaining the pathogenesis of endometriosis.
Improvement of semen quality by nocturnal scrotal cooling and moderate behavioural change to reduce genital heat stress in men with oligoasthenoteratozoospermia
Authors: Jung A , Eberl M , Schill WB
Published: March 2004

A questionnaire assessing factors that might cause an increase in scrotal temperature was completed by patients with reproducible oligoasthenoteratozoospermia of idiopathic nature or caused by varicocele. Evaluation by means of a grading scale revealed increased scrotal heat stress in oligoasthenoteratozoospermic patients compared with normozoospermic men (P < 0.01). In addition, long-term determination of 24 h scrotal temperature profiles showed that compared with semen donors, oligoasthenoteratozoospermic patients frequently had scrotal temperatures above 35.5 degrees C despite the same environmental temperatures (P < 0.05). In 88% of cases, maximum scrotal temperatures were measured during rest or sleep phases, whereas minimum values were recorded during physical activity or frequent change of position. Nocturnal scrotal cooling by means of an air stream resulted in a decrease in scrotal temperature of approximately 1 degrees C. Furthermore, a highly significant increase in sperm concentration (P < 0.0001) and total sperm output (P < 0.0001) was achieved after nocturnal scrotal cooling for 12 weeks together with a moderate decrease in factors leading to genital heat stress. A significant improvement in sperm motility (P < 0.05) and sperm morphology (P < 0.05) was also observed, but this improvement was markedly less pronounced than the changes in sperm concentration. This study shows the importance of genital heat stress as a cofactor in fertility impairment in men and indicates nocturnal scrotal cooling as a therapeutic option.
Effects of arginine- and lysine-vasopressin on phospholipase C activity, intracellular calcium concentration and prostaglandin F2alpha secretion in pig endometrial cells
Authors: Braileanu GT , Simasko SM , Hu J , Assiri A , Mirando MA
Published: March 2004

Oxytocin and vasopressin are related peptides that have receptors in the uterus. Species from families other than Suidae produce only arginine-vasopressin; in contrast, pigs apparently express both arginine- and lysine-vasopressin. The aim of this study was to determine whether arginine- or lysine-vasopressin would activate phospholipase C, increase intracellular calcium concentration [Ca(2+)](i) and stimulate PGF(2alpha) production in enriched cultures of stromal, glandular epithelial and luminal epithelial cells from pig endometrium. Cells were obtained from gilts on day 16 after oestrus by differential enzymatic digestion and sieve separation. After 96 h in culture, the cells were treated with 0 or 100 nmol arginine- or lysine-vasopressin l(-1). The responses to 100 nmol oxytocin l(-1) and 100 nmol GnRH l(-1) were used as positive and negative controls, respectively. Consistent with previous results, oxytocin stimulated phospholipase C activity (P < 0.05), increased [Ca(2+)](i) (P < 0.05) and promoted PGF(2alpha) secretion (P < 0.05) from stromal and glandular epithelial cells. Activity of phospholipase C, [Ca(2+)](i) and PGF(2alpha) release were also increased (P < 0.05) by arginine-vasopressin in stromal cells, but the responses were less (P < 0.01) than those induced by oxytocin. An oxytocin antagonist attenuated the [Ca(2+)](i) response of stromal cells to both oxytocin and arginine-vasopressin. Sequential treatment of cells with oxytocin and arginine-vasopressin indicated that oxytocin desensitized the response to oxytocin, but arginine-vasopressin did not similarly desensitize the response to oxytocin. In glandular and luminal epithelial cells, arginine-vasopressin did not stimulate phospholipase C activity, [Ca(2+)](i) or PGF(2alpha) secretion. Neither GnRH nor lysine-vasopressin induced phospholipase C activity, increased [Ca(2+)](i) or stimulated PGF(2alpha) production in any endometrial cell type. These results indicate that oxytocin receptors can bind arginine-vasopressin more readily than they bind lysine-vasopressin. Type 1 vasopressin receptors may also exist in endometrium predominantly on cells other than stromal, glandular epithelial and luminal epithelial cells, as in previous studies both arginine-vasopressin and lysine-vasopressin stimulated phospholipase C activity in endometrial explants to a similar extent as oxytocin.
Cellular basis for paracrine regulation of ovarian follicle development
Authors: Albertini DF , Combelles CM , Benecchi E , Carabatsos MJ
Published: March 2004

Paracrine factors secreted by oocytes and somatic cells regulate many important aspects of early ovarian follicle development in mammals. From activation of dormant primordial follicles to selection of secondary follicles, locally acting factors have been identified that appear to exert important effects on the growth and differentiation of oocytes and granulosa cells. This article summarizes evidence to support a model for bi-directional paracrine communication that is based on developmental regulation of the delivery and reception of paracrine factors at the oocyte-granulosa cell interface. Transzonal projections that originate from granulosa cells and terminate at the oocyte plasma membrane provide a polarized means to orient the secretory organelles of somatic cells. Characterization of transzonal projections in follicles from normal and genetically modified mice reveals dynamic changes in the density and stability of transzonal projections. On the basis of new data analysing the orientation and cytoskeletal content of transzonal projections in mammalian oocytes, a model is proposed for regulation of paracrine growth factor secretion by follicle-stimulating hormone. These findings have immediate implications for ovarian hyperstimulation protocols and follicle culture models as related to the production of mammalian embryos by assisted reproductive technologies.
Mechanisms for pattern formation leading to axis formation and lineage allocation in mammals: a marsupial perspective
Authors: Selwood L
Published: March 2004

Developing patterns in early embryogenesis are analysed in conceptuses from several families, including Dasyuridae, Phalangeridae, Macropodidae and Didelphidae, in which cleavage has been examined in some detail. Features common to cleavage and blastocyst formation, and in some cases to hypoblast formation, are used to develop an outline of possible mechanisms leading to axis formation and lineage allocation. Relevant features that have been described only in some species are also included. It is suggested that certain features of marsupial cleavage establish patterns in the developing blastocyst epithelia, pluriblast, trophoblast and hypoblast that contribute to axis formation and lineage allocation. All marsupials examined had a polarized oocyte or conceptus, the polarity of which was related to the conceptus embryonic-abembryonic axis and, eventually, the conceptus dorsal-ventral axis and the formation of the pluriblast (future embryo) and trophoblast. The embryonic dorsal-ventral and anterior-posterior axes were established after the allocation of hypoblast and epiblast. Mechanisms that appear to result in patterning of the developing epithelia leading to axis formation and lineage allocation are discussed, and include sperm entry point, gravity, conceptus polarity, differentials in cell-zona, cell-cell and cell-type (boundary effects) contacts, cell division order during cleavage and signals external to the conceptus. A model of the patterning effects is included. The applicability of these mechanisms to other amniotes, including eutherian mammals, is also examined.
Mammalian aromatases
Authors: Conley A , Hinshelwood M
Published: March 2004

Aromatase is the enzyme complex that catalyses the synthesis of oestrogens from androgens, and therefore it has unique potential to influence the physiological balance between the sex steroid hormones. Both aromatase cytochrome P450 (P450arom) and NADPH-cytochrome P450 reductase (reductase), the two essential components of the enzyme complex, are highly conserved among mammals and vertebrates. Aromatase expression occurs in the gonads and brain, and is essential for reproductive development and fertility. Of interest are the complex mechanisms involving alternative promoter utilization that have evolved to control tissue-specific expression in these tissues. In addition, in a number of species, including humans, expression of aromatase has a broader tissue distribution, including placenta, adipose and bone. The relevance of oestrogen synthesis and possibly androgen metabolism in these peripheral sites of expression is now becoming clear from studies in P450arom knockout (ArKO) mice and from genetic defects recognized recently in both men and women. Important species differences in the physiological roles of aromatase expression are also likely to emerge, despite the highly conserved nature of the enzyme system. The identification of functionally distinct, tissue-specific isozymes of P450arom in at least one mammal, pigs, and several species of fish indicates that there are additional subtle, but physiologically significant, species-specific roles for aromatase. Comparative studies of mammalian and other vertebrate aromatases will expand understanding of the role played by this ancient enzyme system in the evolution of reproduction and the adaptive influence of oestrogen synthesis on general health and well being.
Variation in antral follicle development during the follicular phase of the oestrous cycle in red deer (Cervus elaphus) hinds
Authors: McLeod BJ , Meikle LM , Fisher MW , Manley TR , Heath DA , McNatty KP
Published: March 2004

The aim of this study was to quantify antral follicle populations in cyclic red deer hinds and to monitor follicle development leading to ovulation. Oestrus was synchronized with exogenous progesterone and ovaries were recovered approximately 0, 12, 24 or 36 h (follicular phase) or 10 days (luteal phase) after progesterone withdrawal (n = 5 per group). All follicles > or = 2 mm in diameter were dissected out, health status was assessed, follicular fluid oestradiol content was measured, granulosa cells were harvested and their capacity for oestradiol and cAMP production was determined. The time of oestrus and the preovulatory LH surge were monitored in five control hinds. Deer ovaries contained 26.6 +/- 3.45 (mean +/- SEM) follicles > or = 2 mm in diameter (range 4-81), with at least one large antral follicle (diameter: 8.3 +/- 0.38 mm) per hind. There was a strong correlation between follicle size and granulosa cell population (r(2) = 0.676). Approximately half (50.7%) of the follicles were classified as healthy, with the percentage classified as atretic decreasing with increasing follicle size. Neither the total number of antral follicles nor their size distribution differed significantly among groups. There were significantly more (P < 0.05) healthy follicles at 24 h after progesterone withdrawal than at 0 h, when large oestrogenic follicles had fewer granulosa cells, lower follicular fluid oestradiol concentrations and lower aromatase activity (P < 0.05) than did those from other groups. In summary, antral follicle development in red deer is similar to that in other monovulatory ruminants, and at least one large follicle is present at all stages of the oestrous cycle.
Effect of immunization against the alpha N (alphaN) and alpha C (alphaC) peptides of the alpha43 subunit of inhibin on antral follicular growth and atresia and the patterns of gonadotrophin secretion in ewes
Authors: Dhar A , Doughton BW , Pruysers E , Brown RW , Findlay JK
Published: March 2004

The aims of this study were to investigate the role of inhibin in the distribution of healthy and atretic antral follicles and the secretion patterns of gonadotrophins. Ewes were actively immunized against either alphaN or alphaC of the inhibin alpha subunit with a primary injection and three booster injections. The control ewes received adjuvant only. The ovaries were removed either before or at 24 h after hCG administration in a synchronized follicular phase 48 h after removal of intravaginal progesterone pessaries. Morphological observations were made on every fifth section of the complete ovary (one per ewe) stained with haematoxylin and eosin. The mean number of corpora lutea observed per ewe with corpora lutea was not significantly different in ewes immunized against alphaN (2.4; alphaN-immunized ewes) or alphaC (2.6; alphaC-immunized ewes), and control (2.4) ewes, although some corpora lutea appeared cystic in the immunized ovaries. Compared with luteal phase concentrations, mean basal FSH concentrations in the early follicular phase were significantly increased in the alphaC-immunized ewes, similar in alphaN-immunized ewes and reduced in control ewes. No differences were observed in any of the LH parameters. Before hCG treatment, healthy antral follicles > 1 mm in diameter were not observed in any of the 52 follicles in the aC-immunized ewes and were observed in one of 37 follicles from alphaN-immunized ewes compared with 19 of 28 follicles in control ewes (P < 0.0001). For healthy antral follicles < 1 mm in diameter, there were 72 of 85 follicles in the alphaC-immunized ewes, 79 of 81 follicles in the alphaN-immunized ewes and 81 of 82 follicles in the control ewes. Similar results were obtained in healthy antral follicles < 1 mm in diameter at 24 h after hCG administration. In contrast to the control ewes, no healthy preovulatory follicles (> 6 mm in diameter) were observed in alphaN- and alphaC-immunized ewes either before or 24 h after hCG administration. Two newly formed corpora lutea from alphaC-immunized ovaries contained retained oocytes compared with none in control and alphaN-immunized ovaries. In conclusion, immunization against alphaN and alphaC may result in disruption of the normal processes of antral follicular growth and maturation independent of the concentrations of FSH and LH.
Characterization of the active site of monkey sperm hyaluronidase
Authors: Yudin AI , Li MW , Robertson KR , Cherr GN , Overstreet JW
Published: March 2004

The mammalian sperm hyaluronidase, PH-20, is active in macaque spermatozoa at neutral and acid pH. Antibodies were produced to synthesized peptides representing regions of PH-20 that may be involved in hyaluronidase activity and designated peptide 1 (amino acid sequence 142-172) and peptide 3 (amino acid sequence 277-297). Western blotting of proteins extracted from the surface of acrosome-intact spermatozoa showed that the two peptide-specific, affinity-purified IgGs label a 64 kDa band corresponding to the PH-20 molecule. Western blots of acrosome-reacted spermatozoa showed that, under reducing conditions, the two anti-peptide IgGs label the 44 kDa band only, which represents the N-terminal fragment of PH-20. Anti-peptide 3 IgG also labels the 53 kDa form of PH-20 in extracts of acrosome-reacted spermatozoa. Peptide-specific, affinity-purified Fab fragments from both IgGs were shown by fluorescence microscopy and transmission electron microscopy to label the sperm plasma membrane, fused acrosomal vesicles, acrosomal matrix and inner acrosomal membrane. Fab fragments of anti-peptide 1 IgG, but not anti-peptide 3 IgG, inhibited hyaluronidase activity of PH-20 from the sperm surface and from extracts of acrosome-reacted spermatozoa at pH 7.0. Fab fragments of both anti-peptide IgGs inhibited sperm hyaluronidase activity at pH 5.0. It is concluded that the region of PH-20 encompassed by the amino acid sequence 142-172 is essential for hyaluronidase activity at neutral pH, whereas the region of amino acid sequence 277-297 may be more important at a lower pH. It is likely that these two regions are the acid/base catalyst site and the nucleophilic site, respectively, of PH-20 hyaluronidases.
Delayed effect of heat stress on steroid production in medium-sized and preovulatory bovine follicles
Authors: Roth Z , Meidan R , Shaham-Albalancy A , Braw-Tal R , Wolfenson D
Published: March 2004

During the autumn, the conception rate of dairy cattle in warm countries is low although ambient temperatures have decreased and cows are no longer exposed to summer thermal stress, indicating that there may be a delayed effect of heat stress on cattle fertility. Two experiments were conducted to examine possible delayed effects of heat stress on follicular characteristics and steroid production at two distinct stages of follicular growth: medium-sized and preovulatory follicles, 20 and 26 days after heat exposure, respectively. Lactating cows were subjected to heat stress for 12 h a day in an environmental chamber, during days 2-6 of a synchronized oestrous cycle. In Expt 1, ovaries were collected on day 3 of the subsequent cycle, before selection of the dominant follicle, and medium-sized follicles were classified as atretic or healthy. In Expt 2, on day 7 of the subsequent cycle, PGF(2a) was administered and preovulatory follicles were collected 40 h later. In both experiments, follicular fluid was aspirated, granulosa and thecal cells were incubated, and steroid production was determined. In healthy medium-sized follicles (Expt 1), oestradiol production by granulosa cells and androstenedione production by thecal cells were lower (P < 0.05) and the concentration of progesterone in the follicular fluid was higher in cows that had been previously heat-stressed than in control cows (P < 0.05). In preovulatory follicles (Expt 2), the viability of granulosa cells was lower (P < 0.05) and the concentration of androstenedione in the follicular fluid and its production by thecal cells were lower (P < 0.05) in cows that had been previously heat-stressed than in control cows. In both experiments, the oestradiol concentrations in the follicular fluids were not altered by heat stress. These results demonstrate a delayed effect of heat stress on steroid production and follicular characteristics in both medium-sized and preovulatory follicles; this effect could be related to the low fertility of cattle in the autumn.
Mechanisms of cytokine-induced death of cultured bovine luteal cells
Authors: Petroff MG , Petroff BK , Pate JL
Published: March 2004

Tumour necrosis factor alpha (TNF-alpha) and gamma-interferon (IFN-gamma) are cytotoxic to bovine luteal cells in vitro and may contribute to cell death during luteolysis in vivo. In this study, the mechanism by which luteal cells are killed by TNF-alpha and IFN-gamma was investigated. Luteal cells were cultured for 7 days in the presence or absence of TNF-alpha and IFN-gamma. Inhibitors of arachidonate metabolism or scavengers of free radicals were included in the culture media. In addition, the effect of IFN-beta on the viability of cytokine-treated luteal cells was tested. Lastly, untreated and cytokine-treated cells were subjected to single cell gel electrophoresis for quantification of DNA fragmentation. Neither indomethacin nor nordihydroguaiaretic acid, which are inhibitors of cyclooxygenase and lipoxygenase, respectively, were able to prevent cytokine-induced cell death. Similarly, both the phospholipase A(2) inhibitor arachidonyltrifluoromethyl ketone and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine, were largely without effect. In contrast, while vitamin C did not significantly affect viability, superoxide dismutase plus catalase increased viability of cytokine-treated cells (P < 0.05), and IFN-beta prevented cell death (P < 0.05). Finally, while control cells remained free of DNA damage, TNF-alpha plus IFN-gamma induced significant amounts of DNA damage by 48 h after initiation of treatment (P < 0.05). In conclusion, reactive oxygen species, but not arachidonate metabolism or nitric oxide, contribute to cytokine-induced luteal cell death in vitro, and the process of cell death may be via apoptosis. Furthermore, IFN-beta may confer protective effects against cytokine-induced cell death in bovine luteal cells.
Role of prostaglandins in intrauterine migration of the equine conceptus
Authors: Stout TA , Allen WR
Published: March 2004

Between at least day 9 and day 16 after ovulation the spherical equine conceptus migrates continuously throughout the uterine lumen, propelled by peristaltic myometrial contractions. This unusually long period of intrauterine movement ensures that the conceptus delivers its anti-luteolytic signal to the entire endometrium to achieve luteostasis. The present experiment tested the hypothesis that prostaglandins stimulate the myometrial contractions that result in the migration of the conceptus. Serial ultrasonographic examinations of the uteri of eight mares performed during 2 h periods between day 10 and day 18 of gestation recorded the pattern of conceptus migration before and after treatment with the cyclo-oxygenase inhibitor flunixin meglumine. Conceptus mobility was high between day 10 and day 14 after ovulation (4.3 +/- 0.8, 4.7 +/- 0.8 and 4.3 +/- 0.9 changes of location per h on day 10, day 12 and day 14, respectively), but was reduced immediately and markedly by an i.v. injection of flunixin meglumine (3.8 +/- 1.5, 1.8 +/- 0.8 and 0.7 +/- 0.2 location changes per h), thereby implicating prostaglandins as the primary stimulus for the myometrial contractions that drive migration of the conceptus.
Appearance and number of follicles and change in the concentration of serum FSH in female bovine fetuses
Authors: Tanaka Y , Nakada K , Moriyoshi M , Sawamukai Y
Published: March 2004

The aim of this study was to determine the relationships between early follicular development, the time at which follicles appear in early stages of follicular development, and changes in the serum concentrations of FSH in the female bovine fetus. Thirty-five female bovine fetuses aged between 59 and 285 days, as estimated from the crown-rump length, were obtained from dams at an abattoir. Serum samples were separated from fetal blood obtained from the heart. Fetal ovaries were removed and weighed. The content of oestradiol in one of the fetal ovaries and the serum concentrations of FSH and oestradiol were determined using radioimmunoassay. Sections of the other ovary were examined histologically for the appearance and number of follicles. The follicles were divided into four stages: primordial, primary, secondary and early antral. The appearance of primordial, primary, secondary and early antral follicles was observed at day 74, day 91, day 120 and day 150, respectively. Serum concentrations of FSH in female bovine fetuses increased between day 120 and day 150 of gestation. Fetal serum concentrations of oestradiol increased from day 120. The number of early antral follicles increased from day 180 together with an increase in the fetal ovarian content of oestradiol. These findings indicate that, in the female bovine fetus as well as in adult cows, the number of follicles and stages of follicular development are associated with changes in the concentration of FSH.
Different types of oestrous cycle in two closely related South American rodents (Cavia aperea and Galea musteloides) with different social and mating systems
Authors: Touma C , Palme R , Sachser N
Published: March 2004

A comparative approach was used to investigate two closely related South American rodent species: the wild cavy (Cavia aperea) and the yellow-toothed cavy (Galea musteloides). These species of wild guinea-pig inhabit different habitats and show divergent social and mating systems. Cavia have a polygynous mating system, whereas in Galea promiscuous mating occurs. These observations correspond to functional variations in sperm characteristics, which might be interpreted as adaptations to different reproductive patterns in the females of both species. Twenty-two female C. aperea and G. musteloides (11 of each species) were investigated to elucidate differences in the characteristics of the oestrous cycles. The experimental design consisted of three stages, in which the degree of contact between the sexes was successively changed. During these stages, the reproductive cycle of the females was monitored by examining the condition of the vaginal closure membrane, analysing vaginal smears and determining serum concentrations of oestrogens and progesterone. The results revealed different types of oestrous cycle in the two species. Female C. aperea showed periodical cycles with spontaneously occurring oestrous periods, ovulation and corpus luteum activity. In contrast, in female G. musteloides, oestrus was exclusively induced by the presence of a male. However, after the induction of oestrus, ovulation and corpus luteum activity occurred spontaneously without stimuli from copulation. To date this type of oestrous cycle has not been described for any other mammalian species. Thus, these data indicate that differences in the reproductive patterns of the females may have shaped the evolution of different sperm characteristics in the males.
Spermatogonia: stem cells with a great perspective
Authors: Meachem S , von Schonfeldt V , Schlatt S
Published: March 2004

Interest in spermatogonia has grown in recent years as a result of exciting developments in stem cell research in general and the development of new research tools allowing the isolation, culture and transplantation of these cells. This review focuses on the methodological breakthroughs and highlights the recent findings that have substantially increased understanding of spermatogonial physiology. The article provides a comprehensive overview of the hormonal regulation of spermatogonia and presents several new approaches to the use of spermatogonia in basic science and medicine. In the near future these techniques will allow the development of novel routes for the generation of transgenic lifestock, the treatment of infertility, the targeting for male contraception, and an alternative strategy for fertility preservation of oncological patients.
Actions of gonadotrophins on the uterus
Authors: Shemesh M
Published: March 2004

Binding sites for LH/hCG are found in the uterus of several species, including humans. In cattle and pigs, the LH receptor, its mRNA and LH receptor protein are present in the uterus throughout the oestrous cycle, and maximum expression occurs at the luteal phase. GnRH receptor is also expressed maximally in the human endometrium at the luteal phase. LH activates both the adenylate cyclase and phospholipase C pathways and increases the concentrations of cyclooxygenase and its products. Activation of LH receptors in the endometrium is associated with PGF production. In contrast, bovine uterine vein LH receptor mRNA and LH receptor concentrations are greatest during pro-oestrus-oestrus and LH increases the production of both PGE and PGF. FSH receptor and its mRNA are present in the bovine cervix and the concentrations are greatest at the time of the FSH peak value in the blood, indicating a physiological role for FSH in the relaxation and opening of the cervix. The presence of gonadotrophin and releasing factor receptors with a dynamic pattern in the endometrium, myometrium, oviduct and cervix of different species provides evidence that gonadotrophins and GnRH play a substantial role as molecular autocrine-paracrine regulators of the oestrous cycle and implantation.
Temporal changes in reproductive hormones and conceptus-endometrial interactions during embryonic diapause and reactivation of the blastocyst in European roe deer (Capreolus capreolus)
Authors: Lambert RT , Ashworth CJ , Beattie L , Gebbie FE , Hutchinson JS , Kyle DJ , Racey PA
Published: March 2004

The roe deer blastocyst is in diapause between August and December, after which time it expands and elongates rapidly before implantation. Blood samples were taken from 30 animals to define temporal changes in reproductively important hormones to investigate the physiological cues present at embryo reactivation. In 15 of these animals, changes in uterine and conceptus protein synthesis and secretion, and luteal progesterone release during diapause and reactivation, were assessed after culture of these tissues in vitro. Oestradiol concentrations remained low during diapause (1.07 +/- 0.4 pg ml(-1)) and expansion (1.2 +/- 0.4 pg ml(-1)) but increased by 30 times at trophoblast elongation (49.17 +/- 0.37 pg ml(-1)). Prolactin remained at basal concentrations (4.69 +/- 0.86 ng ml(-1)) and increased after implantation (12.34 +/- 2.71 ng ml(-1)). Peripheral progesterone concentrations and luteal progesterone release remained constant throughout diapause, reactivation and implantation (peripheral progesterone: 3.82 +/- 1.97 ng ml(-1); luteal progesterone: 6.72 +/- 0.81 ng mg(-1) protein). Incorporation of a radiolabel into conceptus secretory proteins increased by four times at expansion compared with diapause, whereas incorporation into endometrial secretions remained constant. At elongation, incorporation into endometrial secretions increased two times and conceptus secretions increased 32 times. Two-dimensional electrophoresis and fluorography showed that the profile of endometrial secretory proteins was constant until implantation when qualitative changes were evident. Although a role for an endocrine maternal trigger of reactivation from diapause cannot be dismissed, these data provide no supporting evidence and indicate that the conceptus itself may drive reactivation.
Studies on the metabolism of essential fatty acids in isolated human testicular cells
Authors: Retterstol K , Haugen TB , Tran TN , Christophersen BO
Published: March 2004

The essential fatty acid 22:6(n-3) is a minor component of the Western diet, but a major fatty acid in human testis and semen. In mature spermatozoa, the physical and fusogenic properties of the plasma membrane are probably influenced by its particular fatty acid composition. In this study, the synthesis of 22:6(n-3) and 22:5(n-6) was investigated in isolated human testicular cells. [1-(14)C]20:4(n-6), [1-(14)C]20:5(n-3), [1-(14)C]22:4(n-6) and [1-(14)C]22:5(n-3) were incubated in a 'crude' cell suspension (consisting of a mixture of the cells in the seminiferous tubule), and in fractionated pachytene spermatocytes and round spermatids. The esterification of fatty acids in lipid and phospholipid classes and the fatty acid chain elongation and desaturation were measured. The crude cell suspension metabolized the fatty acids more actively than did the fractionated germ cell suspension, indicating that types of cell other than the germ cells are important for fatty acid elongation and desaturation and thus the production of 22:6(n-3). This finding is in agreement with previous results in rats that indicated that the Sertoli cells are the most important type of cell for the metabolism of essential fatty acids in the testis. Some [1-(14)C]20:5(n-3) was elongated to [(14)C]22:5(n-3) in the fractionated germ cells, but very little was elongated further to [(14)C]24:5(n-3),possibly restricting the formation of [(14)C]22:6(n-3). In the fractionated germ cells, the fatty acid substrates were recovered primarily in the phospholipid fraction, indicating an incorporation in the membranes, whereas in the crude cells, more substrates were esterified in the triacylglycerol fraction. In the phospholipids, more radioactivity was recovered in phosphatidylcholine than in phosphatidylethanolamine and more radioactivity was recovered in phosphatidylethanolamine than in phosphatidylinositol or phosphatidylserine.
Selective sperm binding to pig oviductal epithelium in vitro
Authors: Petrunkina AM , Gehlhaar R , Drommer W , Waberski D , Topfer-Petersen E
Published: March 2004

The sperm reservoir in the caudal isthmus of the oviduct of a number of species is created by binding of spermatozoa to oviductal epithelium. The sperm reservoir fulfills a number of functions such as control of sperm transport, maintenance of sperm viability and modulation of capacitation. The initial capacities of ejaculated and epididymal boar spermatozoa to bind to oviductal epithelium were investigated using a modified pig oviductal explant assay. The number of spermatozoa that bound to 0.01 mm(2) of explant surface was used as the parameter of binding capacity. Binding of spermatozoa to oviductal epithelial explants was dependent in a linear manner on the number of spermatozoa added (P < or = 0.05). No difference was found in initial sperm binding between isthmic and ampullar explants. There was no effect of the stage of the oestrous cycle or the reproductive status of the female donor. There was a significant effect (P < or = 0.05) of the individual boar on the binding index. The binding index correlated negatively with the percentage of spermatozoa with cytoplasmic droplets and the percentage of morphologically abnormal spermatozoa (P < or = 0.05). Epididymal spermatozoa showed significantly lower initial binding capability than did ejaculated spermatozoa from the same boars (P < or = 0.05); therefore, components of seminal plasma may play a role in the binding process. The individual differences revealed by this study and their relation to morphology and contact of spermatozoa with seminal fluid indicate a selective function of sperm-oviduct binding.
Effect of number and diameter of follicles on plasma concentrations of inhibin and FSH in mares
Authors: Donadeu FX , Ginther OJ
Published: March 2004

The role of the number of follicles and circulating immunoreactive inhibin in the decrease in plasma FSH concentrations that occurs during development of a follicular wave was studied in mares. All follicles > or = 6 mm in diameter were ablated by ultrasound-guided transvaginal aspiration of follicular fluid on day 10 after ovulation. During the subsequent wave, all follicles, the three largest follicles (three follicle group), the largest follicle (single follicle group) or no follicles were retained and the remaining follicles were ablated before they reached > 10 mm in diameter (n = 10-11 mares per group). Ablation of new follicles was continued until the day on which the largest follicle of the new wave reached 25 mm in diameter (day 18 after ovulation in the 'no follicle' group). Diameters of retained follicles were measured once a day by transrectal ultrasonography. Plasma samples were taken once a day and analysed by radioimmunoassay for concentrations of FSH and immunoreactive inhibin (includes dimeric inhibin as well as free alpha-subunit forms). Data were normalized to the day of the expected start of the decrease in plasma FSH concentrations (day 0: largest follicle 13 mm in diameter in the follicle-retained groups). A simultaneous increase in circulating concentrations of FSH (P < 0.05) and immunoreactive inhibin (P < 0.05) occurred before the largest follicle reached 13 mm in diameter, which indicates that immunoreactive inhibin produced by follicles < 13 mm in diameter did not suppress FSH. Plasma concentrations of FSH decreased (P < 0.05) and immunoreactive inhibin concentrations increased (P < 0.05) after day 0 in the follicle-retained groups. A slower decrease in FSH concentrations was associated temporally with a delay in the increase in immunoreactive inhibin concentrations in the 'single follicle' group relative to the 'three follicle' and 'all follicle' groups. All follicle-retained groups had similar plasma concentrations of FSH and immunoreactive inhibin after the expected beginning of deviation in growth rates between the two largest follicles (largest follicle 22-23 mm in diameter). These results indicated that the decrease in plasma FSH concentrations from the start of the decrease until the expected day of deviation was a function of multiple follicles of a wave and was attributable to the secretion of inhibin. Thereafter, the largest follicle alone accounted for the continued FSH suppression.