Reproduction

Diamond, M. P.; Moley, K. H.; Pellicer, A.; Vaughn, W. K.; DeCherney, A. H.

doi: 10.1530/jrf.0.0860001pmid: 2526873

Summary. Mice were made diabetic by intraperitoneal injection of streptozotocin or alloxan. Germinal vesicle breakdown in the ovarian follicles at 8 h after hCG in control animals (57%) was significantly greater than in streptozotocin-(24%) and alloxan-(42%) diabetic animals (P < 0·001). This delay in oocyte maturation was reversible by in-vivo insulin administration to diabetic mice. A developmental delay was also found for embryos recovered from diabetic mice. This developmental delay extended into the 72 h in-vitro cultures. Compared to control embryos, those from alloxan- and streptozotocin-treated mice demonstrated marked impairment in development as assessed by (1) distribution of developmental cell stages at each observation period and (2) rates of development which increasingly diverged at each observation period.In diabetic mice treated with insulin in vivo, the percentage of 2-cell embryos recovered increased. Furthermore, in streptozotocin- and alloxan-animals treated with insulin, the rate of in-vitro development of embryos, as well as their developmental stage distribution improved. We therefore suggest that uncontrolled diabetes mellitus, as well as contributing to the development of congenital malformations, may deleteriously affect reproductive performance both before fertilization and at the very earliest gestational stages.Keywords: diabetes mellitus; zygote; oocyte; alloxan; streptozotocin; mouse

Hoffmann, Anna-Maria; Bergh, A.; Olivecrona, T.

doi: 10.1530/jrf.0.0860011pmid: 2569038

Summary. A simple and reliable method was developed to determine the neutral cholesteryl ester hydrolase (CEH) activity in rat testes, using cholestery1-[1-14C]-oleate as substrate. The activity was due to a soluble enzyme present in the cytoplasm of predominantly Sertoli cells, which could be shown after depleting the testes of Leydig cells with ethane dimethyl sulphonate. This treatment also revealed that the loss of CEH activity in abdominal testes of experimentally cryptorchid rats takes place in the Sertoli cells. In prepubertal rats made unilaterally cryptorchid at birth, the CEH activity was significantly higher in the abdominal than in the scrotal testes at 16 days of age. This is earlier than any previously described biochemical change and coincides with, or may even precede, the earliest morphological changes which are accumulation of lipid droplets in the Sertoli cells. The testicular CEH activity then decreased to 30 days of age in the abdominal testes, whereas the activity increased in the contralateral, scrotal testes. When adult rats were made unilaterally cryptorchid for 24 h, the CEH activity decreased rapidly in the abdominal testes. These results suggest that a derangement in cholesteryl ester metabolism is an early event in the pathogenesis of testicular degeneration in cryptorchidism.Keywords: testis; Sertoli cell; cryptorchidism; enzyme activity; assay; ethane dimethyl sulphonate

Gallagher, G. R.; Senger, P. L.

doi: 10.1530/jrf.0.0860019pmid: 2754638

Summary. In Exp. I, virgin Holstein heifers (N = 18) were induced into oestrus with PGF-2α. Animals which stood to be mounted were paired for insemination ∼8 h later with 56·1 × 106 spermatozoa from a single bull. Semen was deposited in the uterine body of one female. Each matched female was inseminated by deposition of one-half of the inseminate into the right uterine horn and one-half into the left uterine horn ∼ 7·0 cm anterior to the internal cervical os. In Exp. II, additional heifers (N = 18) were induced into oestrus and inseminated by deposition into the uterine horns or cervix (2·0 cm anterior to the external cervical os). A 1·0 ml aspirate of vaginal mucus was collected at hourly intervals for 8 h after insemination. Concentration of spermatozoa was determined by haemocytometry. In Exp. I, cumulative percentage spermatozoa recovered in an 8 h collection period were similar (P > 0·10) for insemination into the uterine horns (17·9 ± 2·9%) and uterine body (18·5 ± 4·5%). In Exp. II, cumulative % sperm recovery from the vagina was greater (P < 0·10) for cervical deposition (59-1 ± 14·1%) than for that into the uterine horns (30·9 ± 7·8%). In Exp. II, the insemination treatment × hour of sample interaction was significant (P < 0·08). Recovery of spermatozoa from the vagina was greatest (P < 0·05) within 3 h after cervical insemination (31 ·4 ± 9·9% compared to 9·4 ± 2·5% for uterine horn deposition). Percentage recovery of spermatozoa from the remaining hourly collections were similar (P > 0·10). These results suggest that retrograde movement of spermatozoa from the uterus to the vagina was similar after insemination into the uterine horns or uterine body and 2-fold greater after cervical deposition.Keywords: cattle; insemination site; sperm loss

McNatty, K. P.; Lun, S.; Heath, D. A.; Hudson, N. L.; O'Keeffe, L. E.; Henderson, K. M.

doi: 10.1530/jrf.0.0860027pmid: 2502619

Summary. At 37°C 125I-labelled human (h) FSH (NIAMDD-hFSH-I–3) bound rapidly to granulosa cells from Booroola and Romney ewes with 50% maximum binding achieved after 3 min and equilibrium being reached within 45 min, irrespective of whether the cells were obtained from the FF, F+ or ++ Booroola genotypes or from Romney ewes. Binding of 125I-labelled FSH followed second order kinetics and there was no effect of follicle diameter (1–2·5 mm vs ≥3 mm). Irrespective of breed, genotype or follicle size, the mean (±s.e.m.) calculated association rate constant, (ka) was 7·3 (± 0·8) × 105 litres mol−1 sec−1 (n = 12). Dissociation of receptor bound 125I-labelled hFSH was < 5% after 30 min and low but variable (i.e. between 0 and 30%) after 2–6 h irrespective of breed, genotype or follicle size. No gene-specific differences were noted in binding specificity between F+ and ++ genotypes: studies were not performed with cells from FF ewes because of insufficient cells. The binding of 125I-labelled hFSH could be displaced with sheep FSH (NIH-FSH-S16; 10% cross-reaction) and FSH-P (2·5% cross-reaction) but other sheep pituitary hormones and hCG showed little or no cross-reaction (⩽ 0·1%).The calculated binding capacities (Bmax) and equilibrium dissociation constants (Kd) for 125I-labelled hFSH binding to granulosa cells did not differ between the Booroola genotypes or between Booroola or Romney follicles of different diameter (i.e. 1–2·5 mm; or ≥ 3 mm). The overall mean ± s.e.m. (n = 24) Bmax and Kd values were 16·7 ± 0·8 fm/mg protein (i.e. ∼800 available receptor binding sites/cell) and 1·1 ± 0·1 nm respectively.Collectively, these findings suggest that the earlier maturation of follicles in FF or F + ewes compared to ++ ewes is unlikely to be due to gene-specific differences in the FSH binding characteristics of the granulosa cells.Keywords: Booroola ewes; FSH binding; granulosa cells

Legrand, C.; Banuelos-Nevarez, A.; Maltier, J. P.

doi: 10.1530/jrf.0.0860039pmid: 2754655

Summary. In the early pregnant rat, electrical activity of the myometrium consisted of regular bursts of spike potential, which appeared well propagated on Day 2 of pregnancy. During Day 3, there was a gradual disappearance of propagated activity. Concomitantly, there was a 7-fold increase (P < 0·001) of uterine progesterone concentrations. At this stage, mean duration of bursts was 15·2 ± 0·9 sec and intervals of complete quiescence between bursts were 84·2 ± 7·0 sec. At 10:00 h on Day 4, there were peaks in the uterine concentrations of oestradiol and progesterone, + 36% and + 654%, respectively, compared with values on Day 2 (P < 0·05). Between 10:00 and 20:00 h on Day 4, EMG activity exhibited a rapid and transient rise: bursts were of longer duration at the utero-tubal end of the horn (+ 60%, P < 0·05) with an increased amplitude of spike potentials (+67% and +90% respectively at the tubal and cervical ends of the uterus, P < 0·05). The administration of prazosin depressed EMG activity reversibly in a dose-dependent manner with maximal inhibition at about 2–3 h later. It is concluded that the changes observed during EMG recordings are relevant to the intrauterine distribution of blastocysts and related to changes in the steroidal environment and/or to catecholamine effects via α1-adrenoceptors.Keywords: myometrial activity; blastocyst distribution; prazosin; rat

Isahakia, M. A.

doi: 10.1530/jrf.0.0860051pmid: 2666651

Summary. A monoclonal antibody (BSA6) was generated against an antigenic determinant secreted by the epididymis of the baboon and present on the acrosomal surface of the spermatozoa. This determinant was first secreted by the principal cells of the proximal corpus region, as determined by fluorescent microscopy performed on Bouinfixed epididymal tissue sections. The secretory product subsequently bound on the lateral acrosomal surfaces in the distal corpus region, but became uniformly distributed over the acrosomal region in the cauda epididymidis. The antigenic determinant had a molecular weight of 82 000 (western blot technique). The testis, caput and other somatic tissues were devoid of the antigen, indicating the restriction of the antigen to spermatozoa and epithelial cells of the corpus epididymidis. Examination of similar tissue from immature baboons indicated that the secretion of this antigen was age-dependent, secretion beginning at about 4 years of age.Keywords: monoclonal antibodies; baboon; epididymis; sperm maturation; acrosome.

Mukhopadhyay, P. K.; Chowdhury, M.

doi: 10.1530/jrf.0.0860059pmid: 2754657

Summary. The mannose/fructose-binding agglutinin from Day 1–7 post coitum (p.c.) rat uteri was purified on Concanavalin A. The specific haemagglutination activity peaked on Days 4 and 5 p.c. and a 1·4-fold increase in the yield was accompanied by a 10–12-fold increase in specific agglutination titre. The mannose-binding affinity of the protein also increased, but the highest fructose-binding affinity was found on Day 1 p.c., which may indicate a role of the protein in fructose concentration for utilization by the spermatozoa. Rats that were pseudopregnant, superovulated and pseudopregnant, and had one uterine horn ligated showed that, although a basal level of the protein was induced by the hormonal milieu, actual stimulation of the protein synthesis occurred in the presence of the fertilized ova.Keywords: agglutinin; affinity chromatography; rat; uterus; pregnancy; blastocyst

Kim, J.; Kim, H. K.; Kim, K.; Kim, S. R.; Cho, W. K.

doi: 10.1530/jrf.0.0860065pmid: 2754658

Summary. Mouse embryos were extracted with 0·5% Triton X-100 and subjected to cellulose acetate electrophoresis. In fertilized eggs, two forms of alkaline phosphatase (ALP), a slow-moving form and a fast-moving form, were observed. As cleavage proceeded, the fast-moving form disappeared, and the slow-moving form, the mobility of which was similar to that of the slow-moving form of the kidney, became gradually dominant up to the blastocyst stage (named 'embryonic' form). With blastulation, another fast-moving form showing a similar mobility to the lung ALP began to appear in blastocysts and showed a transient dominance in hatched blastocysts. After implantation, both the embryonic form and the fast-moving form gradually faded, and were eventually replaced by the new form, which may be named 'fetal form' in Day 7 embryos.These results clearly demonstrated that ALP activity does exist in embryos at all stages of preimplantation development. Moreover, the changes in multiple forms of ALP correlated with embryonic development may suggest that these multiple forms may have differential roles in the process of early development.Keywords: alkaline phosphatase; isoenzymes; preimplantation mouse embryo; cellulose acetate electrophoresis

Riley, S. C.; Poyser, N. L.

doi: 10.1530/jrf.0.0860073pmid: 2754659

Summary. The outputs of prostaglandin (PG) F-2α, PGE-2 and 6-keto-PGF-1α from Day-7 and Day-15 guinea-pig endometrium in culture were reduced by the inclusion of actinomycin D, cycloheximide and puromycin in the culture medium, with the output of PGF-2α from Day-15 endometrium being particularly affected during the first 6 h of culture. The intrauterine administration of actinomycin D on Day 10 decreased the outputs of PGF-2α and PGE-2, but not of 6-keto-PGF-1α, from Day-15 endometrium in culture without affecting PG output from Day-15 myometrium in culture. Actinomycin D, cycloheximide and puromycin did not reduce PG output when superfused over the Day-7 and Day-15 guinea-pig uterus in vitro for 20 min, indicating that these compounds do not have a rapid inhibitory effect on endometrial PG synthesis. In fact, they tended to stimulate PG output during this 20-min period, with cycloheximide having a pronounced effect on PGE-2 output.The synthesis of secreted proteins, but not of cellular proteins, was greater by Day-15 than by Day-7 endometrium in culture. Actinomycin D, cycloheximide and puromycin inhibited the synthesis of secreted and cellular proteins by Day-7 and Day-15 endometrium in culture. Protein synthesis and PG synthesis in the endometrium were both inhibited to a greater extent by cycloheximide and puromycin than by actinomycin D. The intrauterine administration of actinomycin D on Day 10 reduced the syntheses of secreted and cellular proteins by Day-15 endometrium in culture. These findings indicate that the endometrial synthesis of PGs, particularly of PGF-2α towards the end of the oestrous cycle, is dependent upon endometrial protein synthesis.Keywords: guinea-pig; uterus; prostaglandins; protein synthesis

Gustafson, A. W.; Damassa, D. A.; Pratt, R. D.; Kwiecinski, G. G.

doi: 10.1530/jrf.0.0860091pmid: 2502624

Summary. The binding of sex steroids to plasma proteins was examined in post-natal Djungarian and golden hamsters. With dihydrotestosterone or testosterone as ligands, steady-state polyacrylamide gel electrophoresis revealed 2 androgen binding components in the plasma of young Djungarian hamsters of both sexes. The fast-moving component exhibited a low affinity and high capacity for androgens and corresponded to albumin in stained gels. In contrast, the slow moving component was a β-globulin with high affinity (Ka = 109 m −1) and low capacity for androgens, and was identified as a specific sex steroid-binding protein (SBP; also SHBG). This SBP did not bind oestrogens or corticosteroids and was electrophoretically distinct from corticosteroid-binding globulin (CBG). In addition, this protein did not appear to be of testicular origin because it was present in immature females and in immature males that had been castrated for 8 days. Plasma concentrations of SBP in males as measured by a diethylaminoethyl-cellulose binding assay were low at birth, became significantly elevated shortly thereafter when plasma androgen values were also elevated, and subsequently fell to low levels during puberty. These changes follow the same general pattern that has been described for other mammals, including humans, during this period of reproductive development. Although the significance of elevated SBP concentrations during prepuberty has yet to be determined, it appears that the increased concentrations of high-affinity androgen binding in the plasma of Djungarian hamsters plays a role in the asynchronous pubertal development of the testes and accessory organs which occurs in this species. The post-natal SBP pattern in females was similar to that observed in males. Plasma SBP levels were low or undetectable in adults of both sexes. As previously described for adult golden hamsters, the plasma of post-natal male and female golden hamsters lacked a specific SBP: androgen binding in this species is apparently limited to albumin and CBG.Keywords: SBP/SHBG; CBG; steroid-binding; puberty; Djungarian hamster; golden hamster