Reproduction

Aitken, R. John

doi: 10.1530/jrf.0.1150001pmid: 10341716

A great deal of evidence has accumulated in recent years to suggest that there has been a gradual increase in male reproductive pathology over the past 30–40 years, as evidenced by increased rates of testicular cancer and declining semen quality. The hypothesis is advanced that this phenomenon is causally related to the ability of male germ cells to generate reactive oxygen metabolites. When produced in low levels, such metabolites are thought to enhance sperm function by stimulating DNA compaction and promoting a redox-regulated cAMP-mediated pathway that is central to the induction of sperm capacitation. When produced in excessive amounts, the same metabolites stimulate DNA fragmentation and a loss of sperm function associated with peroxidative damage to the sperm plasma membrane. Free radical-induced mutations in the male germ line may also be involved in the aetiology of childhood cancer and recent increases in the incidence of seminoma. In light of these considerations, establishing the mechanisms for free radical generation by the male germ line and determining the factors that influence this activity are important objectives for future research in this area.

Akazome, Y.; Mori, T.

doi: 10.1530/jrf.0.1150009pmid: 10341717

In chicken embryos, there is a difference between the sexes in the onset of lutropin receptor mRNA expression in the gonads. The effects of oestrogen on lutropin receptor expression were studied to investigate the mechanism controlling this difference. Lutropin receptor mRNA expression was detected in the ovaries of sesame oil-treated control female embryos on day 12 of incubation, while no expression was found in the testes of the male controls. Oestradiol administration to genetically male embryos before sexual differentiation resulted in gonadal sex reversal which was characterized histologically by the proliferation of cortical cords and the presence of lacunae. Lutropin receptor expression was detected in the feminizing testis on day 12 of incubation. Administration of aromatase inhibitor (CGS 16949 A) to genetically female embryos before sexual differentiation inhibited the formation of cortical cords, although a relatively weak expression of lutropin receptor was detected. These results indicate that early expression of the lutropin receptor is regulated by oestrogen.

Salfen, B. E.; Cresswell, J. R.; Xu, Z. Z.; Bao, B.; Garverick, H. A.

doi: 10.1530/jrf.0.1150015pmid: 10341718

The presence of a developing dominant follicle may be a factor in the control of the luteolytic cascade mechanism and the number of follicular waves during the bovine oestrous cycle. In this study, ovaries of all animals were examined once a day by transrectal ultrasonography. It was expected that heifers (n = 18) would have two follicular waves if the second wave occurred later than day 10 after oestrus (Expt 1) and that cows (n = 14) would have three waves if the second wave occurred on or before day 10 (Expt 2). The objective of Expt 1 was to determine if absence of a large follicle late in the luteal phase delays luteal regression in heifers that are expected to have two follicular waves. Nine heifers were injected i.v. with 10 ml charcoal-treated bovine follicular fluid three times a day for 4 days, starting on the day after initiation of the second follicular wave, to delay growth of the second wave dominant follicle. Nine heifers were injected with 0.9% NaCI as controls. The duration of the luteal phase (calculated as the number of days that serum progesterone was > 0.5 ng ml−1) was greater (P < 0.01) in the follicular fluid-treated group compared with the controls (18.7 versus 14.1 days). FSH and follicular growth were suppressed during the period of injection of follicular fluid (P < 0.01 and 0.03, respectively). The objective of Expt 2 was to determine the effect of increased oestradiol on the duration of the luteal phase in cows that were expected to have three follicular waves. Seven cows were injected i.m. three times a day for 4 days with 1 ml oestradiol (100 μg ml−1 in corn oil) and seven cows were similarly injected three times a day with 1 ml 0.9% NaCI (control) starting the day after cessation of growth of the second wave dominant follicle. Luteal phase duration was shorter in oestradiol-treated animals than in the controls (14.0 versus 19.0 days; P < 0.04). Serum oestradiol concentrations were higher in the oestradiol-treated group during the period of injection (P < 0.01). In summary, luteolysis was delayed when follicular growth was suppressed with follicular fluid (Expt 1). Exogenous oestradiol administration during the development of uterine oestradiol responsiveness initiated luteolysis earlier compared with control animals (Expt 2).

Oliveira, R. G.; Tomasi, L.; Rovasio, R. A.; Giojalas, L. C.

doi: 10.1530/jrf.0.1150023pmid: 10341719

The dynamic parameters of mouse sperm cells exposed to follicular and oviductal fluids were assessed. Spermatozoa were tracked on a chemotactic Zigmond chamber and recorded using a videomicroscopy system. The results were evaluated with computer-supported image analysis. Follicular fluid at a dilution of 10−4 markedly increased the proportion of spermatozoa with high velocity, and stimulated chemotactic behaviour. The highest velocities were observed in sperm cells exposed to oviductal fluid, and a greater proportion of these cells had high velocity compared with those exposed to follicular fluid. Chemotaxis was induced in spermatozoa exposed to oviductal fluid at dilutions of 10−3 and 10−5. These results suggest the presence of temporal subpopulations of responsive spermatozoa, considering the distance travelled towards both follicular and oviductal fluids and the proportion of sperm cells migrating towards the gradient in the highest distance ranges. This is the first report on the effect of isolated follicular and oviductal fluids on dynamic parameters and chemotaxis of mouse spermatozoa. The findings support previous work showing that the motility and directionality of mouse sperm cells is increased by factors in the microenvironment of the egg. Although the significance of these factors in vivo is unknown, it is possible that there is a relay mechanism involving sequential activity of both oviductal and follicular fluids to direct the male gametes towards the egg.

Nath, D.; Majumder, G. C.

doi: 10.1530/jrf.0.1150029pmid: 10341720

Highly purified plasma membranes, isolated by an aqueous two-phase polymer method from goat epididymal spermatozoa, were found to possess a kinase activity that causes phosphorylation of serine and threonine residues of several endogenous plasma membrane proteins. Cyclic AMP, cyclic GMP, Ca2+–calmodulin, phosphatidylserine–diolein, polyamines and heparin had no appreciable effect on this kinase. Autoradiographic analysis showed that the profile of the phosphorylation of membrane proteins by this endogenous cAMP-independent protein kinase underwent marked modulation during the transit of spermatozoa through the epididymis. In caput sperm plasma membrane, 18, 21, 43, 52, 74 and 90 kDa proteins were phosphorylated, whereas, in the corpus and cauda epididymal spermatozoa, a differential phosphorylation pattern was observed with respect to the 90, 74, 21 and 18 kDa proteins. The rate of phosphorylation of the 74 kDa protein decreased markedly during the early phase of sperm maturation (caput to distal corpus epididymides) whereas there was little change in kinase activity in sperm plasma membrane. In contrast, the rates of phosphorylation of the 18 and 21 kDa proteins increased during the terminal phase (distal corpus to distal cauda epididymides) of sperm maturity, although the kinase activity of membrane decreased significantly during this phase. The modulation of the phosphorylated states of these specific membrane proteins may play an important role in the maturation of epididymal spermatozoa.

Kot, K.; Ginther, O. J.

doi: 10.1530/jrf.0.1150039pmid: 10341721

The characteristics of follicle evacuation during ovulation and the development of the corpus luteum until day 5 (day 0 = ovulation) were studied in seven nulliparous Holstein heifers using real-time ultrasonography. Ovulation was induced and synchronized with a single injection of PGF2α followed in 36 h by GnRH. Continuous scanning and videotaping was performed from apparent stigma formation until antral fluid was no longer detected. The beginning of follicular evacuation (second 0) was defined, retrospectively, after the antral area decreased 10% or more in 1 s. The completion of evacuation was defined as the inability to detect the antrum (the beginning of luteal development, 0 h). Corpora lutea development was monitored at 0, 4 and 20 h, and every 24 h thereafter until day 5. Changes in the maximal cross-sectional area of the antrum, luteal tissue, and central luteal cavities and in the pixel intensity of luteal tissue were determined using a computerized image program. The initial antral fluid evacuation occurred in two patterns that could be readily separated: (1) rapid, means of 58 and 89% evacuation in 1 and 4 s, respectively (four heifers); and (2) slow, means of 17 and 35% in 1 and 4 s, respectively (three heifers). The initial loss that distinguished the two patterns involved about 4 and 20 s for rapid and slow evacuation, respectively. Thereafter, the loss patterns were similar for the two types. The time from the beginning to the completion of evacuation ranged from 6 s to 14.5 min. Mean luteal tissue area increased (P < 0.05) between completion of evacuation (91.2 ± 6.5 mm2) and day 3 (164.4 ± 13.7 mm2) and between day 3 and day 4 (263.4 ± 26.6 mm2). The growth rate of the luteal tissue area between day 3 and day 4 (103.2 ± 16.0 mm2 day−1) was greater (P < 0.05) than that between day 2 and day 3 (41.9 ± 12.4 mm2 day−1) and between day 4 and day 5 (49.7 ± 22.0 mm2 day−1). In contrast to increasing luteal tissue area, mean pixel intensity decreased (P < 0.05) progressively between the completion of evacuation (78.4 ± 6.3) and day 2 (60.4 ± 2.5) and did not change significantly thereafter. In conclusion, initial follicular fluid loss during ovulation occurred in two patterns, involving about 4 and 20 s, respectively. The most intensive luteal tissue growth occurred between day 3 and day 4, and the echogenicity of the luteal tissue decreased between day 0 and day 2.

Driancourt, M. A.; Guet, P.; Reynaud, K.; Chadli, A.; Catelli, M. G.

doi: 10.1530/jrf.0.1150045pmid: 10341722

In cattle, it has been suggested that follicular fluid has direct modulatory effects on follicular growth and maturation. In the first part of this study, an in vitro test using aromatase activity of follicular wall fragments as an end point was validated for cattle follicles and was used to test whether follicular fluid (from dominant or non-dominant follicles) modulates aromatase activity. Fluid from dominant follicles at a concentration of 24 or 12% (obtained during the luteal and follicular phases, respectively) significantly inhibited aromatase activity. Inhibitory activity was low or absent in fluid from non-dominant follicles. FSH-stimulated aromatase activity was also reduced by fluid from dominant follicles, but not to a greater extent than in basal conditions. Finally, charcoal-treated fluid from dominant follicles retained its inhibitory activity. In contrast, ovarian venous serum draining a dominant follicle had no activity at the three concentrations tested (6, 12 and 24%). In the second part of the study, identification of the compounds involved in this modulatory activity was attempted using SDS-PAGE. Comparison of the fluorographs from de novo synthesized proteins stored in follicular fluid (inhibitory medium) with those secreted in incubation medium (inactive medium) demonstrated that one protein (90 kDa, pI 5.8) was significantly (P < 0.05) more abundant in fluid from dominant follicles (2.0 ± 0.09%) than in the culture medium (1.3 ± 0.1% of the total proteins). This protein had characteristics similar to those of heat shock protein 90 (hsp 90). Therefore, in the final part of the study, the presence of hsp 90 in ovarian cells and follicular fluid was investigated using immunohistochemistry and western blot analysis. After immunohistochemistry, a positive signal was detected mainly in the granulosa cells of larger follicles and to a smaller extent in thecal cells and oocytes. Western blot analysis also demonstrated the presence of hsp 90 in follicular wall fragments and fluid. When blotting was achieved on a sample of follicular fluid resolved by two-dimensional PAGE, the spot detected had a similar location to that at 90 kDa and pI 5.8. Addition of purified hsp to bovine follicles in vitro depressed aroaromatase altering the ltalue (and Kmossibly the poss value) oVmaxe enzyme. It is proposed that rop 90 is a functional regulator of follicular maturation through its action on aromatase.

Demmers, K. J.; Kaluz, S.; Deakin, D. W.; Jabbour, H. N.; Flint, A. P. F.

doi: 10.1530/jrf.0.1150059pmid: 10341723

A type I interferon secreted by early sheep and cow conceptuses is responsible for the maternal recognition of pregnancy in these species. Interferon-tau (IFNτ) acts locally on the maternal endometrium to prevent luteolysis and prolong progesterone secretion. The production of IFNτ was investigated in early pregnancy in red deer, Cervus elaphus. The oestrous cycles of 14 hinds were synchronized using intra-vaginal controlled internal progesterone-releasing devices. Hinds were run with a fertile stag, then slaughtered on either day 20 (n = 10) or day 22 after withdrawal of progesterone (n = 11). Conceptuses were recovered after uterine excision and flushing with sterile saline. Conceptus RNA was reverse transcribed and amplified by PCR using primers designed from highly conserved regions of ovine and bovine IFNτ genes. The resulting PCR products were cloned and fully sequenced. Sequence comparisons indicate that the transcript characterized is closely related to the IFNτ and interferon-omega genes of bovids and giraffe, showing > 85% nucleotide sequence homology and > 74% predicted amino acid similarity with previously cloned genes. Northern blot analysis of total conceptus RNA using a homologous IFNτ probe confirmed the high expression of IFNτ which is encoded by a transcript of approximately 1 kilobase. Anti-viral activity was measured in uterine flushes from pregnant hinds using a cytopathic effect inhibition assay (4.3 × 103 ± 0.78 × 103 iu ml−1; n = 14), but was not detectable in flushes from nonpregnant hinds (n = 7), confirming that preimplantation red deer conceptuses release interferons. This is the first demonstration of IFNτ expression in a cervid conceptus and provides evidence that IFNτ may be involved in the maternal recognition of pregnancy in red deer.

Picton, H. M.; Campbell, B. K.; Hunter, M. G.

doi: 10.1530/jrf.0.1150067pmid: 10341724

Studies were carried out to investigate the conditions required for maintenance of aromatase activity and expression in long-term cultures of pig granulosa cells. Cells from large (> 2 mm) and small (≤ 2 mm) follicles were cultured at 37°C with 5% CO2 in McCoys 5a medium supplemented with 0.1% (w/v) BSA, testosterone (100 μg l−1), insulin (10 μg l−1) and long R3 insulin-like growth factor I (IGF-I) (100 μg l−1). Cells were cultured with five concentrations of USDA pFSH-I-2 (0–100 μg l−1) for 48, 96 or 144 h with or without fetal calf serum (FCS). The number of cells and oestradiol, progesterone and inhibin production were measured. In marked contrast to oestradiol production from cells cultured in plates precoated with FCS, 1 μg FSH l−1 was optimal for the maintenance of high oestradiol production by granulosa cells from large follicles after 144 h of serum-free culture. Culture with FCS promoted cell proliferation, reduced oestradiol production, and supported FSH-dependent (P < 0.01) increased progesterone and inhibin production indicating cellular luteinization. Northern blot analysis of total RNA from cells cultured with 1 μg FSH l−1 detected 2.5 and 1.8 kb transcripts encoding aromatase cytochrome P450 (P450arom) and cholesterol side-chain cleavage cytochrome P450 (P450scc), respectively. Transcript expression was hormone sensitive, irrespective of the presence of FCS. High concentrations of FSH (100 μg l−1) stimulated expression of P450scc, but inhibited P450arom expression as the cells luteinized after 144 h of culture. This serum-free system, which maintains the aromatase enzyme complex, is fundamental if physiologically relevant observations are to be made of the mechanisms regulating follicle hierarchy development from long-term cultures of pig cells.

Spindler, R. E.; Renfree, M. B.; Shaw, G.; Gardner, D. K.

doi: 10.1530/jrf.0.1150079pmid: 10341725

The tammar wallaby, Macropus eugenii, has a ruminant-like digestive system which may make a significant concentration of amino acids and fatty acids available to the blastocyst via uterine fluids. Fluorescent and radioisotope analyses were performed to determine the rate of glutamine and palmitate use by blastocysts recovered on day 0, 3, 4, 5 and 10 after reactivation induced by removal of pouch young (RPY). Between day 0 and 4 glutamine uptake increased from 15.6 ± 6.6 to 36.1 ± 2.7 pmol per embryo h−1 (P < 0.01) and ammonium production increased from 8.2 ± 4.3 to 26.6 ± 3.0 pmol per embryo h−1 (P < 0.01). Glutamine oxidation did not increase until day 10 after RPY (P < 0.01), but the percentage of glutamine oxidized increased from 4.5 ± 3.1% during diapause to 31.2 ± 12.6% (P < 0.01) by day 5 after RPY and increased further to 51.0 ± 15.8% (P < 0.01) by day 10 after RPY. Palmitate oxidation also increased from 0.3 ± 0.1 by day 0 blastocysts to 3.8 ± 1.7 pmol per embryo h−1 (P < 0.01) by day 4 blastocysts. This increase provides a greater potential for ATP production, possibly to supply increased demand due to the coincident resumption of mitoses. The ATP:ADP ratio within blastocysts had reduced by the time of the first measurement at day 3 (0.5 ± 0.2 pmol per embryo h−1; P < 0.01) compared with day 0 blastocysts (1.4 ± 0.3 pmol per embryo h−1). It is likely that metabolism of amino acids and fatty acids contributes to the energy supply during reactivation of tammar wallaby blastocysts after embryonic diapause.