Showing papers in "Reproduction in 1984"

Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics

Abstract: The objective of this study was to develop a relatively simple test to evaluate the functional integrity of the membranes of human spermatozoa. As in some other species, human spermatozoa 'swell' under hypo-osmotic conditions due to the influx of water and the expansion of the membranes. A mixture of equal parts of fructose and sodium citrate (150 mosmol) with calculated ionic strength of 0.15 resulted in a maximal number of clearly identifiable swollen spermatozoa. Only small variations were seen when different aliquants of the same semen samples were separately evaluated. A high correlation (r = 0.94) was obtained between expected and observed values of swollen spermatozoa when known amounts of heat-treated spermatozoa, unable to undergo swelling, were added to untreated spermatozoa. A good correlation (r = 0.90) was also observed between the % spermatozoa in a semen sample that were capable of undergoing swelling and the % of denuded hamster oocytes that were penetrated by capacitated spermatozoa from the same semen sample. By contrast, the correlations between % sperm swelling in ejaculates and % normal sperm forms, % motile spermatozoa and % spermatozoa that do not stain with eosin-Y (supravital stain) in the same ejaculates were 0.30, 0.61 and 0.52, respectively. Therefore, the hypoosmotic swelling technique to evaluate the functional integrity of the sperm membrane appears to give high repeatability and accuracy and is closely correlated to the in-vitro fertilizing ability of spermatozoa. It may be a useful addition to the standard semen analysis.

Pyruvate and glucose uptake by mouse ova and preimplantation embryos

Abstract: A method has been devised to measure the uptake of nutrients by small numbers of mouse ova and preimplantation embryos. The 1-5 embryos were incubated as a group at 37 degrees C in a small volume (at least 20 nl/embryo) of culture medium on siliconized microscope slides under mineral oil. Serial 4 nl samples were taken at 30-min intervals for 3 h and assayed for their content of glucose and pyruvate using an ultramicrofluorometric technique. Pyruvate uptake exceeded that of glucose in unfertilized and fertilized ova and in the developmental stages up to the blastocyst, when glucose became the predominant substrate. A component of the uptake of pyruvate by unfertilized ova was probably mediated by a carrier.

Pulsatile secretion of gonadotrophins, ovarian steroids and ovarian oxytocin during the periovulatory phase of the oestrous cycle in the cow.

Abstract: An injection of 500 micrograms prostaglandin (PG) analogue was given on Day 12 (mid-luteal phase) of the oestrous cycle to 8 cows. An LH surge occurred 59 +/- 2 h later. LH, FSH, prolactin, oestradiol-17 beta, progesterone and oxytocin concentrations were determined in blood samples collected from the caudal vena cava and/or the jugular vein at 20-min (5 cows) or 5-min (3 cows; only LH and FSH concentrations were determined) intervals for 24 h, beginning 48 h after the PG injection. Oxytocin concentrations were low and similar in the vena cava and the jugular vein. In blood samples collected every 5 min the interpulse interval for LH and FSH during the period before the LH surge was 38-40 min. In the 20-min samples the interpulse interval for oestradiol was similar to that for LH and FSH, but pulse amplitude and basal concentrations steadily increased to reach maximum concentrations 6-8 h before and again during the LH surge. A decrease in oestradiol concentrations, lasting at least 60 min, occurred just before the start of the LH surge. Progesterone concentrations also increased at the same time as the LH surge. The magnitude of the LH surges varied from 7 to 32 ng/ml, but all cows ovulated and had oestrous cycles of normal length. Distinct pulses of LH and FSH were observed throughout the LH and FSH surges. Pulsatile secretion of LH was not detected for a period of up to 6-12 h following the LH surge, but then low-amplitude pulses occurred. In contrast, the pulsatile secretion of FSH remained at a frequency similar to that observed during the descending phase of the FSH surge. Furthermore, a second increase in FSH concentrations occurred, beginning 4-12 h after the LH-surge. It is concluded that: (1) the frequent, high-amplitude pulses of oestradiol that occur before and during the LH surge are probably due to stimulation by pulses of LH; (2) the LH surge is the result of an increase in frequency and amplitude of the LH pulses; (3) the second increase in FSH that follows the LH and FSH surges appears to be the result of an increase in the amplitude (not frequency) of the FSH pulses; and (4) very little, if any, oxytocin is secreted from the ovary during the periovulatory phase of the oestrous cycle.

Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles

Abstract: Early bovine embryos (1- to 8-cell stages) were recovered from superovulated heifers at slaughter on Days 2 or 3. Embryos were cultured for 3-4 days in Medium B2 supplemented with 15% (v/v) fetal calf serum in the absence (B2SS, 106 embryos) or presence of trophoblastic vesicles (B2SS + TV, 190 embryos). At the end of culture, there were more (P less than 0.001) morulae (greater than or equal to 16 cells) in B2SS X TV (46%) than in B2SS alone (18%) irrespective of the initial cell stage. More 8-cell embryos reached the 16-cell stage than did embryos with less than 8 cells (30% vs 15% in B2SS, P greater than 0.05; 70% vs 41% in B2SS + TV, P less than 0.005). After culture, 102 morulae were transferred non-surgically to temporary recipient heifers (84 embryos cultured in B2SS + TV and 18 in B2SS). After 2 or 3 days, 14 out of 58 embryos from the B2SS + TV group and 3 out of 10 embryos from the B2SS group were recovered as blastocysts. Most blastocysts were deep-frozen and stored for several weeks. After thawing, 10 apparently normal embryos from the B2SS + TV group were transferred non-surgically into 10 recipient heifers. Four pregnancies were induced, but only one embryo survived to term (birth of a normal male calf). It is concluded that trophoblastic vesicles release one or several unknown compound(s) normally present in vivo, promoting the cleavage of early bovine embryos.

Some aspects of thecal and granulosa cell function during follicular development in the bovine ovary

Abstract: The patterns of ovarian follicular development and the steroidogenic properties of individual follicles (greater than or equal to 2 mm diam.) were assessed in Angus cows from Day - 5 until Day + 1 of the oestrous cycle (oestrus = Day 0). Individual follicles were judged to be healthy or atretic using a new classification system incorporating assessments of thecal vascularity and colour, the number of granulosa cells, the presence or absence of debris in follicular fluid and the status of the oocyte. The results suggest that the theca interna of small antral follicles (less than 5 mm diam.) responds to LH and synthesizes androstenedione before the granulosa cells develop an appreciable ability to metabolize androgen to oestrogen. Regardless of follicle size, the output of thecal androstenedione per unit mass of tissue remained unchanged in healthy but not in atretic follicles. On a per cell basis, aromatase activity increased in granulosa cells from healthy but not from atretic follicles with increasing follicle size. Peak levels of aromatizing activity were consistently observed in dominant oestrogen-enriched follicles on Day 0 although similar activity was also observed in some healthy follicles (greater than or equal to 8 mm diam.) on other days of the cycle. Early atresia in bovine follicles was characterized by an absence or lowering of aromatase activity in granulosa cells which always preceded any reduction in the thecal steroidogenic response to LH. It was estimated that between 20 and 60 antral follicles (greater than or equal to 2 mm diam.) per cow may respond to LH by synthesizing androgen whereas only 1-3 follicles (greater than 5 mm diam.) have granulosa cells capable of metabolizing androstenedione or testosterone to oestradiol.

Meiotic competence in vitro of pig oocytes isolated from early antral follicles.

Abstract: Pig oocytes were isolated from early antral follicles of different sizes and their abilities to resume and complete meiotic maturation in vitro were compared. After 24 h of culture, more than 80% of the oocytes from follicles 0.3-0.7 mm in diameter remained at the germinal vesicle stage, while 66, 94.3 and 100% oocytes from follicles 0.8-1.6, 1.7-2.2 and 3-5 mm in diameter, respectively, completed germinal vesicle breakdown. After 48 h of culture, 35% of the oocytes in the smallest follicle class progressed to prometaphase and only 4% to metaphase I. Of the oocytes from follicles 0.8-1.6 mm in diameter, 23% reached metaphase I and 17.3% metaphase II. About 50 and 76% of the oocytes from follicles 1.8-2.2 mm and 3-5 mm in diameter, respectively, extruded the first polar body. The ability to resume meiosis (i.e. to undergo germinal vesicle breakdown) is reached by porcine oocytes when they approach their full size in antral follicles greater than 0.8 mm in diameter and before they are capable of completing it (i.e. reaching metaphase II). The ability to complete meiotic maturation acquired in antral follicles of about 2 mm in diameter coincided with a significant decrease in the nucleolar transcriptional activity of the oocytes.

Pre-ovulatory arrest and peri-ovulatory redistribution of competent spermatozoa in the isthmus of the pig oviduct

Abstract: Using the surgical approach of post-coital ligation and transection of the distal oviduct at different times relative to ovulation, together with subsequent recovery of the eggs, gilts mated at the onset of oestrus were studied for progression of viable spermatozoa within the isthmus. Results are derived from 76 animals and examination of 1047 eggs. Transection of the isthmus 1.5-2.0 cm proximal to the utero-tubal junction at intervals from 3 to 36 h after mating prevented fertilization in 269 of 270 eggs, whereas 98% of 223 eggs were fertilized in the control oviducts. Transection at 38 h (pre-ovulatory), 40 h (peri-ovulatory) and 42-44 h (post-ovulatory) after mating yielded, respectively, 5%, 40% and 100% fertilization. The mean number of spermatozoa associated with the zona pellucida increased in a parallel manner. These results, and those obtained with ligatures placed closer to the site of fertilization just after ovulation, indicate a pre-ovulatory arrest of viable spermatozoa in the caudal region of the isthmus for 36 h or more followed by an active ad-ovarian redistribution.

Pulsatile secretion of gonadotrophins, ovarian steroids and ovarian oxytocin during prostaglandin-induced regression of the corpus luteum in the cow

Abstract: Summary. A luteolytic dose (500 \g=m\g)of cloprostenol was given on Day 12 of the oestrous cycle to 5 heifers. Blood samples were collected simultaneously from the caudal vena cava and jugular vein at 5\p=n-\20-minintervals from \p=n-\6 to 0 (control period), 0 to 12 and 24 to 36 h after PG injection. Pulses of LH were secreted concomitantly with pulses of FSH during all sampling periods. However, during the control period separate FSH pulses were detected resulting in a shorter (P < 0\m=.\01)interpulse interval for FSH than LH (93 versus 248 min). LH and FSH pulse frequencies increased (P < 0\m=.\01)beginning 1\p=n-\3h after PG to interpulse intervals of 59 and 63 min, respectively, and continued to be maintained 24\p=n-\36h after PG. Concomitantly there was a 2\p=n-\3-foldincrease (P < 0\m=.\01)in basal concentrations and pulse amplitude for LH (but not FSH). FSH basal concentrations and pulse amplitudes decreased (P < 0\m=.\05)in 3 heifers 24\p=n-\36h after PG. Pulsatile secretion of oestradiol was observed at frequencies similar to LH during the periods 4\p=n-\12h (3 heifers) and 24\p=n-\36h (2 heifers) after PG, respectively, resulting in higher (P < 0\m=.\05)mean oestradiol concentrations. Progesterone concentrations in the vena cava increased (P < 0\m=.\01)5\p=n-\10min after PG but decreased (P < 0\m=.\01)67% by 20 min after PG. This decrease was followed by a rise (P < 0\m=.\05)beginning 2\p=n-\3h after PG and lasting for an average of 3\m=.\3h. After a steady decline, basal concentrations of 1\m=.\0 ng/ml were reached 24\p=n-\36h after PG. Basal oxytocin concentrations in the vena cava and jugular vein (8\m=.\2and 4\m=.\2pg/ml) increased (P < 0\m=.\01)to reach maximum concentrations (2029 and 701 pg/ml) 5\p=n-\10min after PG and then decreased over a 3\p=n-\5h period and were lowest (4\p=n-\3and 3\p=n-\2pg/ml) 24\p=n-\36h after PG. Maximum prolactin concentrations were higher and appeared 5\p=n-\10min earlier in the jugular vein compared to the vena cava. It is concluded that: (1) progesterone and oxytocin secretion from the corpus luteum is initially increased and then dramatically decreased by a luteolytic dose of PG ; (2) the reduction of progesterone concentrations below a certain threshold level in the presence of low oestradiol concentrations probably eliminates the negative feedback effect on gonadotrophin secretion, thereby allowing the frequency and amplitude of LH pulses, and to a lesser extent the frequency of FSH pulses, to increase; and (3) the increase in FSH and LH pulse frequencies probably stimulates, after a variable period of time, the development of a large follicle that secretes increasing concentrations of oestradiol.

Proteins released by cultured Day 15-16 conceptuses prolong luteal maintenance when introduced into the uterine lumen of cyclic ewes.

Abstract: Experiments were performed to determine whether proteins, produced and released into the incubation medium by Day 15-16 sheep conceptuses cultured for 24-48 h, could prolong the functional lifespan of the corpus luteum (CL) when infused into the uterine lumen of cyclic ewes. Beginning on Day 12 (oestrus = Day 0) either a concentrated (2 ml) solution of total conceptus culture medium protein (2.2 mg) or diluted sheep serum (2.2 mg protein) was introduced daily via an indwelling catheter into the uterine lumen of 3 ewes for 7 days (Days 12-18). Peripheral blood samples were collected daily for 14 days (Days 12-25). On Day 25 all ewes were laparotomized and ovaries observed to determine whether CL previously marked with India ink were maintained. All controls had ovulated and formed new CL. By contrast none of the conceptus protein-treated ewes had ovulated, and their peripheral progesterone levels remained elevated. One ewe maintained a functional CL until Day 52 when she was hysterectomized. Light microscopy of histological sections prepared from the endometrium revealed glandular development comparable to that in the endometrium of cyclic animals during late dioestrus. The cells of the CL were similar to those from cyclic animals during mid- to late dioestrus. Ovine trophoblast protein ( oTP -1), a major protein secreted by the sheep conceptus between Days 13 and 21 of pregnancy, was purified from conceptus incubation medium and injected (0.2 mg protein/day) into the uterine lumen of 3 animals. Plasma progesterone concentrations indicated that oTP -1-treated animals maintained luteal function 4 days longer than did control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the elongate spermatid-Sertoli cell ratio in various mammals.

Abstract: Criteria were devised for determining the elongate spermatid-Sertoli cell ratio in various mammalian species at the electron microscope level. When data from particular species were pooled, the values were: rabbit, 12.17:1, hamster, 10.75:1; gerbil, 10.64:1; rat, 10.32:1; guinea-pig, 10.10:1; vole, 9.75:1; and monkey, 5.94:1. The elongate spermatid-Sertoli cell ratio is a measure of the workload of the Sertoli cell and is a prime factor determining their efficiency. The higher the ratio, the higher the sperm output is likely to be per given weight of seminiferous tubule parenchyma for a particular species.

Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators

Abstract: Capacitation of rhesus monkey spermatozoa was assessed by monitoring sperm flagellar beat and trajectory changes during incubation in vitro and by determining sperm penetration into rhesus oocytes and hamster zona-free ova. Rhesus sperm capacitation in vitro depended on the addition to the culture medium of the cyclic nucleotide mediators, caffeine and dibutyryl cyclic AMP. Capacitation was correlated with the development of hyperactivated motility. Spermatozoa treated with the cyclic nucleotide mediators, and showing hyperactivated motility, penetrated 57.4% of all rhesus oocytes and fertilized 88.9% of mature rhesus oocytes that were morphologically normal. Control spermatozoa did not penetrate any of the eggs. Some sperm penetration into hamster ova occurred but was not statistically significant. These data provide a basis for achieving in-vitro fertilization in the rhesus monkey and information on specific sperm motility characteristics associated with fertilizing ability.

Influence of age on sperm production and testicular weights in men

Abstract: Age-related changes in daily sperm production (DSP) and testicular weights were investigated in paired testes from 89 men aged 21-50 years and 43 men aged 51-80 years. For both DSP/testis and DSP/g parenchyma, remarkably large standard deviations exceeded 50% of mean values. However, DSP/g and DSP/testis for both right and left testes were approximately 30% higher in the younger than in the older group (P less than 0.01) and were negatively correlated with age (P less than 0.01) when data from both groups were pooled. Weights of whole testes and of testicular parenchyma were similar in both age groups and were not significantly correlated with age. However, testicular tunic weights were 29% higher in the older group (P less than 0.001) and were positively correlated with age (P less than 0.001). Both testicular tunic weight and the % of total testis occupied by tunic were negatively correlated with DSP/g (P less than 0.01); these correlations were weakened by removing the effect of age. Although total testicular weight and testicular parenchymal weight did not change with age, these values were about 10% lower on the left than on the right (P less than 0.001). In addition to its increase with age, testicular tunic weight was about 8% greater for right than for left testes in all men (P less than 0.001). Although the average size of the testis varied from right to left, DSP/g was similar in paired testes (P = 0.15), and the correlation between right and left DSP/g was high (rho = +0.89, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Maintenance of the corpus luteum after uterine transfer of trophoblastic vesicles to cyclic cows and ewes

Abstract: One or two trophoblastic vesicles (0.4-2 mm diam.) from cow (Day 14) or ewe (Day 11-13) embryos without their disc were transferred, after culture for 24 h, into recipients. Each vesicle was transferred into the uterine horn ipsilateral to the CL by the cervical route in heifers and surgically in ewes on Day 12 of the oestrous cycle. In cows, daily measurements of plasma progesterone concentrations and checks for return to oestrus showed that the CL was maintained in 8 out of 12 recipients. These 8 cows had 25- to 37-day cycles while 4 recipient heifers returned to oestrus normally. Three recipients with an extended cycle were slaughtered. The dissected uterus showed that trophoblastic vesicles had developed in the uterine horns. In ewes, the serum progesterone curve, determined in each recipient, showed that the CL was maintained in 7 out of 12 recipients. These 7 ewes had 20- to 54-day cycles and the other 5 ewes had a normal cycle of 15-19 days comparable to that of 17.0 +/- 0.5 days for the 6 control ewes. Whenever the CL was maintained, high blood progesterone levels were followed by rapid luteolysis. In cattle and sheep, therefore, a trophoblastic vesicle transferred into the uterus can develop in vivo, secreting the embryonic signals when there is no embryonic disc control and transforming the cyclic CL into a CL of pregnancy in about 60% of the cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of an oestrus-associated glycoprotein in oviducal fluid of the sheep.

Abstract: Samples of oviducal fluid were collected daily from sheep with indwelling catheters. Fluid samples taken from both oviducts of 2 sheep for 2 cycles during the middle of the breeding season (April/May) (8 sets of data) were compared with 9 sets of data generated from 2 cycles in 3 sheep later in the breeding season (June/July). Around the period of oestrus, the output of oviducal fluid increased to a peak volume of 1.56 +/- 0.35 ml per day (mean +/- s.d.) compared with a mid-cycle volume of 0.49 +/- 0.29 ml. Later in the breeding season, the flow rates were lower, but showed the same trend (0.91 +/- 0.24 ml at the peak and 0.25 +/- 0.18 ml 7 days later). The total amount of protein secreted by the oviduct each day increased 2-4-fold around the time of oestrus, with higher levels in mid-season ewes. When oviducal fluids were fractionated by SDS electrophoresis, a novel glycoprotein, subunit size of Mr 80-90 000 was identified in samples for 3-6 days of each cycle, coinciding with the period of high fluid flow rate. This protein first appeared in the oviducal fluid on the day of oestrus or the following day and it represented 1 of the 2 major glycoproteins in oviducal fluid as assessed by periodic acid-Schiff (PAS) staining. A PAS-positive protein (Mr 80-90 000) was also detected in fluid taken after oestrus on native highly cross-linked gradient gels after electrophoresis at pH 3.1 but not at pH 8.3. Both gradient gel systems showed an increase in high molecular weight material (Mr greater than 10(6] in fluid taken soon after oestrus.

Effect of sperm concentration during preincubation in a defined medium on fertilization in vitro of pig follicular oocytes.

Abstract: \m=x\108 cells/ml) resulted in a low penetration rate (11%). Epididymal spermatozoa preincubated at a concentration of 4 \m=x\108 cells/ml could also penetrate denuded oocytes. None of the oocytes were penetrated by epididymal spermatozoa that were exposed to seminal plasma before preincubation or by ejaculated spermatozoa. After preincubation, whiplash motility was observed in the epididymal spermatozoa, but not in the ejaculated spermatozoa.

Changes in plasma concentrations of LH, progesterone and oestradiol in relation to the occurrence of luteolysis, oestrus and time of ovulation in the Shiba goat (Capra hircus).

Abstract: Luteolysis in Shiba goats was spontaneous (N = 5) or induced by prostaglandin F-2 alpha (N = 5). Blood sampling and the test for oestrous behaviour were carried out at 2-h intervals, and the time of follicular rupture (ovulation) was determined by culdoscopic observations performed every 2 h around the periovulatory period. In both groups plasma LH concentrations showed a temporary but significant increase during the abrupt fall in plasma progesterone concentrations at luteolysis. This LH rise may be responsible for the preovulatory development of antral follicles and the increase in oestradiol secretion from them. The total number of antral follicles (spontaneous luteolysis 3.6 +/- 0.6, induced luteolysis 4.2 +/- 0.7; mean +/- s.e.m.) and the number of ovulations (1.8 +/- 0.4, 2.4 +/- 0.2) did not differ significantly between the two groups. There was also no significant difference in the timing of oestrus, LH surge and ovulation following the two modes of luteolysis. The interval from luteolysis to the peak of the LH surge averaged 65 h (range 56-72 h). The period of oestrous behaviour coincided with the acrophase of the LH surge and lasted for 22 h (12-28 h). Ovulations occurred 21 (16-24) h after the LH peak and 7 (2-12) h after the end of oestrus.

Comparative protective actions of gonadotrophins and testosterone against the antispermatogenic action of ethane dimethanesulphonate

Abstract: Summary. After a single dose of ethane dimethanesulphonate (EDS) (75 mg/kg) to rats the prolonged antispermatogenic action is due to a temporary elimination of the functional Leydig cell population. Replacement therapy with testosterone propionate (3 mg/day) maintains the spermatogenic epithelium but the EDS effect develops when hormone treatment is discontinued. In contrast, a short treatment with hCG (10\p=n-\100 i.u./day) or LH (714 \g=m\g/day),starting before the EDS dose, permanently protects the spermatogenic epithelium. FSH treatment was completely ineffective. Although histological protection of spermatogenesis appeared complete with testosterone or hCG, effects on fertility remained but over different periods of time. Antispermatogenic and antifertility effects were produced in mice using much higher doses of EDS (5 \m=x\250 mg/kg) but there was no protection from androgen or hCG. It is suggested that EDS binds to Leydig cells irreversibly, interfering with the action of gonadotrophin. At the dose level used the evidence suggests that the degree of reaction renders most of the Leydig cell population non-viable. A direct cytotoxic effect of the compound upon the spermatogenic epithelium might account for the inability of testosterone or hCG alone or in combination to maintain fertility at normal levels.

Mouse sperm capacitation in vitro involves loss of a surface-associated inhibitory component.

Abstract: The increasing fertility of epididymal mouse sperm suspensions as preincubation time is extended is accompanied by the inactivation or destruction of an inhibitory component. Alternatively, precocious removal of the component, achieved by centrifugation, leads to significant improvement in fertilizing ability. Suspensions were preincubated for a total of 120 min, with aliquants being removed at 5, 30 and 120 min. By gently washing samples and resuspending in fresh medium, the poor fertility of unwashed 5- and 30-min suspensions was increased such that 30-min washed samples did not differ significantly from fully capacitated, highly fertile 120-min unwashed control samples. When the supernatants obtained during washing of uncapacitated suspensions (5 and 30 min preincubation) were added to capacitated (120 min preincubation) populations, fertilization of cumulus-intact eggs was markedly and significantly inhibited, although fertilization of zona-free eggs was unaffected. In contrast, supernatants from capacitated suspensions were not inhibitory. When suspensions were preincubated in Ca2+-free media, both washing and exposure to hyperosmolal conditions improved fertilizing ability after addition of exogenous Ca2+, although not to the extent seen in control samples. Removal of the inhibitory component therefore increased the response of spermatozoa to Ca2+. The component was shown to be cell-associated and to inhibit the acrosome reaction in capacitated suspensions. Finally, the inhibition was shown to be reversible, with further incubation of inhibited suspensions restoring the original fertility.

Production of monozygotic (identical) horse twins by embryo micromanipulation.

Abstract: The blastomeres of 192- to 8-cell embryos recovered surgically 1-3 days after ovulation from 23 Pony mares were mechanically separated and inserted, in various combinations, into evacuated pig zonae pellucidae to make 27 'half' and 17 'quarter' micromanipulated embryos. These were embedded in agar and cultured in vivo in the ligated oviducts of ewes for 3.5-5 days to allow development to the late morula/early blastocyst stage. Subsequent surgical or non-surgical transfer of 13 'half' and 17 'quarter' embryos to mares resulted in 10 established pregnancies, including 2 monozygotic pairs. Surgical transfer to mares that had not been recently used as donors of embryos was more successful (10/20) than surgical or non-surgical transfer to recently operated mares (0/10).

Effect of forskolin on the spontaneous maturation and cyclic AMP content of rat oocyte—cumulus complexes

Abstract: Forskolin (0-100 microM), a reversible stimulator of the catalytic subunit of adenylate cyclase, induced dose-response increases in the % germinal vesicle (GV) (ID50 = 2.68 microM, where ID50 is the dose of forskolin which maintained the meiotic arrest at the germinal vesicle stage, determined cytogenetically, of 50% cultured oocytes), and the cAMP content (determined by RIA) of cumulus-enclosed oocytes and cumulus masses. A significant positive correlation was established between the amount of cAMP within the cumulus mass and that in the corresponding enclosed oocyte (r = 0.78). In contrast, neither the % GV nor the cAMP content of cumulus-free oocytes was affected by the drug. The arresting action of forskolin upon cumulus-enclosed oocytes was dependent upon the presence of adherent cumulus cells, and was transient and fully reversible. The gradual decrease in the % GV of cumulus-enclosed oocytes cultured from 4 to 12 h in 25 microM-forskolin (84, 54 and 22% GV at 4, 8 and 12 h, respectively) was accompanied by a drastic fall in intra-oocyte cAMP at 8 h (41.2 +/- 2.5 and 1.1 +/- 0.6 fmol/oocyte at 4 and 8 h, respectively), while the cumulus cell cAMP content remained constant (135.3 +/- 14.7 and 145.9 +/- 28.7 fmol/cumulus at 4 and 8 h, respectively). Moreover, heterologous metabolic coupling, assessed by determination of the fraction of radiolabelled uridine marker that was transferred from the cumulus mass to the oocyte, significantly decreased. These results show that cumulus cell cAMP is transferred to the rat oocyte where it appears to play a pivotal role in the regulation of meiosis and that the rat oolemma does not appear to possess active catalytic subunits of adenylate cyclase in an amount adequate to stimulate measurably cAMP synthesis.

Effects of corticosterone and dietary changes in the hen on ovarian function, plasma LH and steroids and the response to exogenous LH-RH.

Abstract: Ovarian regression was induced in hens by infusing 30 micrograms corticosterone/h, feeding diets deficient in Ca2+ or Na+ and by withdrawal of food and water. The weight of the ovary was most severely reduced by the corticosterone infusion. The total number of normal ovarian follicles weighing greater than 0.012 g was not altered by any of the treatments. However, the number of large yolk-filled follicles decreased while the numbers of smaller follicles and atretic follicles increased when ovarian regression was induced by dietary changes or hormone infusion as compared to normally fed or solvent-infused hens. These experimental treatments resulted in decreases in plasma concentrations of LH, progesterone and oestradiol, and increases in the plasma levels of corticosterone. These changes were immediate except for the low sodium diet with which there was a delay of about 6 days. When fasted birds were fed oats and given water, plasma LH and oestradiol, but not progesterone, increased. The infusion of corticosterone did not affect the ability of the pituitary gland to secrete LH after an injection of LH-RH, but this response was reduced or eliminated by the other experimental treatments. It is concluded that the regression of the ovary induced by these experimental treatments is a consequence of the reduction in the secretion of LH, which may itself be caused by increased plasma levels of corticosterone. It also appears that recruitment of follicles in the maturational stage which precedes entry into the hierarchy of large yolky follicles was unaffected by all of the methods of inducing ovarian regression which were studied.

Endocrine control of antler growth in red deer stags

Abstract: Observations of body weight, testis size, antler status, plasma testosterone and prolactin were made on 12 red deer stags during their first 2 years of life. Six of the stags were fed to appetite throughout the study (Group A) and 6 were fed a 70% restricted diet during each winter (Group B). In addition 6 of the stags , 3 from each group, were studied in more detail; LH and testosterone were measured either after a single injection of LH-RH or in samples taken at frequent intervals over a period of 8 or 24 h. During the study the stags became sexually mature, developed first their pedicles and then antlers and showed at least one complete cycle of casting and regrowth of the antlers . The stags in Group A developed their testes and pedicles about 2 months earlier than did those in Group B. Pedicle initiation was associated with increasing plasma testosterone levels in response to changes in LH secretion, and antler development occurred when testosterone levels were low or decreasing. Cleaning of the velvet was associated with high levels of plasma testosterone. Antler casting occurred when plasma testosterone concentrations were low or undetectable and prolactin levels were high or increasing. The relationship between LH and testosterone varied during the study; in spring when the testes and antlers were growing, relatively high levels of LH were associated with only small peaks of testosterone, yet in summer, when antler growth was complete and the antlers were clean of velvet, low LH concentrations were associated with large peaks of testosterone.

Two-step freezing of two-cell rabbit embryos after partial dehydration at room temperature

Abstract: Summary. The effect of rapid freezing and thawing on the survival of 2-cell rabbit embryos was examined. When embryos in 2\m=.\2m-propanediol were directly plunged from room temperature to liquid nitrogen some of them survived after thawing (8%) but only if they had been pretreated by exposure to an impermeable solute, sucrose, that makes the blastomeres shrink osmotically before cooling. High survival (77\p=n-\88%)in vitro was obtained when pretreated embryos were first held at \p=n-\30\s=deg\Cfor 30\p=n-\240min before immersion into liquid nitrogen. Transfer of such frozen\p=n-\thawedembryos gave a survival rate to live young similar to that obtained with controls (26% and 32% respectively). DMSO was less effective than propanediol; only 2 out of 38 sucrose\x=req-\ pretreated frozen\p=n-\thawedembryos developed in vitro. The present work shows that a combination of partial dehydration of blastomeres at room temperature with their permeation by a cryoprotective agent offers a simple method for successful rapid freezing and thawing of rabbit embryos.

Antibodies against epididymal glycoproteins block fertilizing ability in rat.

Abstract: Antiserum against rat androgen-dependent secretory epididymal protein DE (raised in rabbit) was added to suspensions of rat spermatozoa from the cauda epididymidis which were used for artificial insemination. While control spermatozoa fertilized 41.6% of oocytes, those exposed to antiserum to protein DE fertilized only 6.6% (P less than 0.01). An equal amount of normal rabbit serum (NRS) did not cause inhibition (33.1%). To study the entry of antibodies into the epididymis, caudal tubules were cultured for 24 h and the fertility of the contained spermatozoa was assessed by artificial insemination. Culture in Medium 199 alone or with NRS resulted in spermatozoa which fertilized 52% of oocytes while the presence of antiserum to protein DE in the culture medium yielded spermatozoa which fertilized only 16.6% of oocytes (P less than 0.01). These results suggest (1) that the epididymal protein DE might be part of a sperm structure involved in the fertilization process, and (2) that, at least under the present culture conditions, immunoglobulins penetrate the epididymal epithelium in sufficient numbers to reduce fertility significantly.

Effects of lipid peroxide formation in fowl semen on sperm motility, ATP content and fertilizing ability

Abstract: The ability of samples of semen from individual male fowl to form the products of lipid peroxidation during 5 h aerobic incubation at 40 degrees C varied between 0 and 8 nmol malonaldehyde/10(9) spermatozoa. Formation of higher concentrations of malonaldehyde was associated with a partial or complete loss of fertilizing ability whilst the fertilizing ability of samples producing low or negligible concentrations of malonaldehyde remained unimpaired. The semen of birds which showed a tendency to form high concentrations of malonaldehyde was not readily identifiable as abnormal by assessment of sperm motility, morphology or ATP content. Nor was the loss of fertilizing ability during aerobic incubation associated with an obvious change in these characteristics.

Observations on the response of human spermatozoa to gravity, boundaries and fluid shear

Abstract: Human sperm motility response to three mechanical stimuli, gravity, fluid flow shear and rigid boundaries, was measured in a tube of 310 X 400 microns calibre. Data were gathered by cine recordings at various focussing levels d across the tube and analysed with a computerized image analysis system. The most influential stimulus was the tube wall near (more than 'at') which the swimmers tended to accumulate, leaving the fluid beyond 100 microns from the wall (d = 100) vacant of motile spermatozoa. The boundary effect was evident as soon as the spermatozoa could be viewed after loading, and accumulation, measured as frequency, as a function of d did not change with time t. This response was not significantly altered by the addition of laminar flow with a centre line velocity of about 400 microns/sec. In flow shear, spermatozoa aligned positively (in the flow direction) at the wall but negatively by about 30 microns from the wall where the velocity gradient (= shear rate) was about 3.5 sec-1. The response to gravity was relatively weak with 11 spermatozoa positive (swimming downwards) for each 9 negative. Neither the boundary effect nor the 'rheotaxic' effect were influenced by gravity as there was no statistical difference in orientation or distribution patterns between vertically and horizontally flowing suspensions. It is suggested that the boundary effect cannot be ignored in in-vitro manipulations, particularly when spermatozoa are observed or extracted. Its importance in vivo lies in the degree to which the tubes transporting motile spermatozoa seem to have mechanisms for reversing the wall accumulation tendency.

Origin of anti-Müllerian hormone in bovine freemartin fetuses

Abstract: The origin of AMH responsible for Mullerian duct regression in bovine freemartins has been reinvestigated, using a sensitive RIA for this hormone Between 50 and 80 days, Mullerian duct regression occurs simultaneously in males and freemartins Both twins exhibited high and positively correlated serum AMH concentrations, whereas gonadal in-vitro production of AMH and biological anti-Mullerian activity were detectable at a low level only in 2 out of 13 freemartins In the gonads of approximately half the freemartins after 80 days, seminiferous tubules differentiated and the gonads produced AMH, but the output was very low compared to that of the male twin These data suggest that regression of Mullerian duct in freemartins is essentially mediated by AMH produced by the testes of the male twin