Different patterns of metabolic cryo-damage in domestic cat (Felis catus) and cheetah (Acinonyx jubatus) spermatozoa.

Felid spermatozoa are sensitive to cryopreservation-induced damage, but functional losses can be mitigated by post-thaw swim-up or density gradient processing methods that selectively recover motile or structurally-normal spermatozoa, respectively. Despite the importance of sperm energy production to achieving fertilization, there is little knowledge about the influence of cryopreservation or post-thaw processing on felid sperm metabolism. We conducted a comparative study of domestic cat and cheetah sperm metabolism after cryopreservation and post-thaw processing. We hypothesized that freezing/thawing impairs sperm metabolism and that swim-up, but not density gradient centrifugation, recovers metabolically-normal spermatozoa. Ejaculates were cryopreserved, thawed, and processed by swim-up, Accudenz gradient centrifugation, or conventional washing (representing the 'control'). Sperm glucose and pyruvate uptake, lactate production, motility, and acrosomal integrity were assessed. Mitochondrial membrane potential (MMP) was measured in cat spermatozoa. In both species, lactate production, motility, and acrosomal integrity were reduced in post-thaw, washed samples compared to freshly-collected ejaculates. Glucose uptake was minimal pre- and post-cryopreservation, whereas pyruvate uptake was similar between treatments due to high coefficients of variation. In the cat, swim-up, but not Accudenz processing, recovered spermatozoa with increased lactate production, pyruvate uptake, and motility compared to controls. Although confounded by differences in non-specific fluorescence among processing methods, MMP values within treatments were positively correlated to sperm motility and acrosomal integrity. Cheetah spermatozoa isolated by either selection method exhibited improved motility and/or acrosomal integrity, but remained metabolically compromised. Collectively, findings revealed a metabolically-robust subpopulation of cryopreserved cat, but not cheetah, spermatozoa, recovered by selecting for motility rather than morphology.

Oxidative phosphorylation is essential for felid sperm function, but is substantially lower in cheetah (Acinonyx jubatus) compared to domestic cat (Felis catus) ejaculate.

Compared with the normospermic domestic cat, sperm metabolic function is compromised in the teratospermic cat and cheetah, but the pathway(s) involved in this deficiency are unknown. Glycolysis is essential for sperm motility, yet it appears to function normally in spermatozoa of either species regardless of structural morphology. We conducted a comparative study to further understand the mechanisms of energy production in felid spermatozoa, with the hypothesis that oxidative phosphorylation is required for normal sperm function and is impaired in teratospermic ejaculates. Electroejaculates from both species were stained with MitoTracker to quantify mitochondrial membrane potential (MMP) or were incubated to assess changes in sperm function (motility, acrosomal integrity, and lactate production) after mitochondrial inhibition with myxothiazol. Sperm midpiece dimensions also were quantified. Sperm mitochondrial fluorescence (directly proportional to MMP) was ~95% lower in the cheetah compared with the normospermic and teratospermic cat, despite the cheetah having a 10% longer midpiece. In both species, MMP was increased 5-fold in spermatozoa with retained cytoplasm compared with structurally normal cells. Inhibition of oxidative phosphorylation impaired sperm function in both species, but a 100-fold higher inhibitor concentration was required in the cat compared with the cheetah. Collectively, findings revealed that oxidative phosphorylation was required for sperm function in the domestic cat and cheetah. This pathway of energy production appeared markedly less active in the cheetah, indicating a species-specific vulnerability to mitochondrial dysfunction. The unexpected, cross-species linkage between retained cytoplasmic droplets and elevated MMP may reflect increased concentrations of metabolic enzymes or substrates in these structures.

Glycolytic enzyme activity is essential for domestic cat (Felis catus) and cheetah (Acinonyx jubatus) sperm motility and viability in a sugar-free medium.

We have previously reported a lack of glucose uptake in domestic cat and cheetah spermatozoa, despite observing that these cells produce lactate at rates that correlate positively with sperm function. To elucidate the role of glycolysis in felid sperm energy production, we conducted a comparative study in the domestic cat and cheetah, with the hypothesis that sperm motility and viability are maintained in both species in the absence of glycolytic metabolism and are fueled by endogenous substrates. Washed ejaculates were incubated in chemically defined medium in the presence/absence of glucose and pyruvate. A second set of ejaculates was exposed to a chemical inhibitor of either lactate dehydrogenase (sodium oxamate) or glyceraldehyde-3-phosphate dehydrogenase (alpha-chlorohydrin). Sperm function (motility and acrosomal integrity) and lactate production were assessed, and a subset of spermatozoa was assayed for intracellular glycogen. In both the cat and cheetah, sperm function was maintained without exogenous substrates and following lactate dehydrogenase inhibition. Lactate production occurred in the absence of exogenous hexoses, but only if pyruvate was present. Intracellular glycogen was not detected in spermatozoa from either species. Unexpectedly, glycolytic inhibition by alpha-chlorohydrin resulted in an immediate decline in sperm motility, particularly in the domestic cat. Collectively, our findings reveal an essential role of the glycolytic pathway in felid spermatozoa that is unrelated to hexose metabolism or lactate formation. Instead, glycolytic enzyme activity could be required for the metabolism of endogenous lipid-derived glycerol, with fatty acid oxidation providing the primary energy source in felid spermatozoa.

Evidence for compromised metabolic function and limited glucose uptake in spermatozoa from the teratospermic domestic cat (Felis catus) and cheetah (Acinonyx jubatus).

Cheetahs and certain other felids consistently ejaculate high proportions (≥ 60%) of malformed spermatozoa, a condition known as teratospermia, which is prevalent in humans. Even seemingly normal spermatozoa from domestic cat teratospermic ejaculates have reduced fertilizing capacity. To understand the role of sperm metabolism in this phenomenon, we conducted a comparative study in the normospermic domestic cat versus the teratospermic cat and cheetah with the general hypothesis that sperm metabolic function is impaired in males producing predominantly pleiomorphic spermatozoa. Washed ejaculates were incubated in chemically defined medium containing glucose and pyruvate. Uptake of glucose and pyruvate and production of lactate were assessed using enzyme-linked fluorescence assays. Spermatozoa from domestic cats and cheetahs exhibited similar metabolic profiles, with minimal glucose metabolism and approximately equimolar rates of pyruvate uptake and lactate production. Compared to normospermic counterparts, pyruvate and lactate metabolism were reduced in teratospermic cat and cheetah ejaculates, even when controlling for sperm motility. Rates of pyruvate and lactate (but not glucose) metabolism were correlated positively with sperm motility, acrosomal integrity, and normal morphology. Collectively, our findings reveal that pyruvate uptake and lactate production are reliable, quantitative indicators of sperm quality in these two felid species and that metabolic function is impaired in teratospermic ejaculates. Furthermore, patterns of substrate utilization are conserved between these species, including the unexpected lack of exogenous glucose metabolism. Because glycolysis is required to support sperm motility and capacitation in certain other mammals (including dogs), the activity of this pathway in felid spermatozoa is a target for future investigation.

Zona-free embryo culture: is it a viable option to improve pregnancy rates?

Sporadic reports published during the previous decade have documented pregnancies achieved with transfer of zona-free human embryos. Although the overall efficiency seems to be good and some authors have suggested systematic application for special infertility problems, there have been only a few attempts to compare the benefits of zona-free embryo culture and transfer with the traditional approach using zona-intact embryos. So far, the majority of instances in which zona-free culture has been applied have occurred accidentally. This review summarizes the known functions of the zona pellucida, analyses natural and artificial situations where its function is compromised, including zona hardening and difficult hatching that seem to be related to in-vitro embryo culture, and discusses possible methods and timing for artificial zona removal. With the availability of in-vitro systems capable of replacing important functions of the zona pellucida, routine use of zona-free culture for the whole in-vitro period, after or even before fertilization, is a realistic possibility with potential additional benefits. Based on the increasing amount of animal studies, a systematic comparison is suggested that may eventually diminish the handicaps of the in-vitro situation and lead to simplification of manipulations as well as higher success rates after embryo transfer.

Incidence of chromosomal mosaicism in morphologically normal nonhuman primate preimplantation embryos.

OBJECTIVE: To establish the exact rates of chromosomal mosaicism in morphologically normal rhesus macaque embryos by determining the chromosomal complement of all blastomeres. DESIGN: Retrospective rhesus monkey IVF study. SETTING: Academic laboratory and primate research center. PATIENT(S): Young fertile rhesus macaque females. INTERVENTION(S): Morphologically normal in vitro-produced rhesus macaque embryos were dissociated and cytogenetically assessed using a five-color fluorescent in situ hybridization assay developed for rhesus macaque chromosomes homologous to human chromosomes 13, 16, 18, X, and Y. MAIN OUTCOME MEASURE(S): The incidence and extent of chromosomal mosaicism in rhesus macaque preimplantation embryos. RESULT(S): Seventy-seven preimplantation embryos, displaying normal morphology and development, from 17 young rhesus macaque females were analyzed. Overall, 39 embryos (50.6%) were normal, 14 embryos (18.2%) were completely abnormal, and 24 embryos (31.2%) were mosaic. Of the 226 blastomeres analyzed in the mosaic group, 110 blastomeres (48.7%) were normal. CONCLUSION(S): The observed rate of mosaicism in good-quality rhesus embryos resembles previously documented frequencies in poor-quality human preimplantation embryos. A high incidence of mosaicism may limit the diagnostic accuracy of preimplantation genetic diagnosis.

Effects of in vitro maturation and age on oocyte quality in the rhesus macaque Macaca mulatta.

OBJECTIVE: To evaluate oocyte quality in a primate model. DESIGN: Analysis of oocyte karyotype by chromosome spreading and oocyte spindles by confocal microscopy. SETTING: Research laboratory, Caribbean Primate Research Center. ANIMAL(S): Rhesus macaques aged 6-22 years. INTERVENTION(S): Fourteen females underwent both Regimen A (FSH + hCG) and Regimen B (FSH only) stimulation cycles to facilitate collection of mature and immature oocytes. Immature oocytes from Regimens A and B underwent in vitro maturation (IVM) to produce metaphase II oocytes. All metaphase II oocytes underwent gradual fixation to spread chromosomes or were fixed and stained with probes specific to alpha-tubulin, actin, and DNA for visualization of the meiotic spindle using confocal microscopy. MAIN OUTCOME MEASURE(S): Karyotype and meiotic spindle architecture differences among in vivo matured (IVO) and IVM oocytes from young and old rhesus macaques. RESULT(S): In all, 4.7% of IVO oocytes (Regimen A) from young females were hyperhaploid versus 25.0% of IVM oocytes (Regimen B) from old females; 4.5% of IVO oocytes (Regimen A) from young females versus 51.5% of IVM oocytes (Regimen B) from old females displayed abnormal chromosome alignment on the metaphase spindle. CONCLUSION(S): IVM can induce meiotic anomalies in macaque oocytes, especially those obtained from older females. Results from this study provide possible explanations for the reported reduction in developmental competence of IVM primate oocytes.

Rhesus macaque embryos derived from MI oocytes maturing after retrieval display high rates of chromosomal anomalies.

BACKGROUND: Rhesus macaque and human preimplantation embryos display similar rates of chromosomal abnormalities. The aim of this study was to determine whether embryos developing from MI oocytes that mature post-retrieval display more chromosomal anomalies than those embryos that are generated from oocytes that are at MII at the time of retrieval. METHODS: Rhesus macaque oocytes were obtained after hormonal ovarian stimulation. Immediately after retrieval, the oocytes were classified according to their maturational status. Following in vitro fertilization, Day 3 embryos with good morphology and development derived from oocytes maturing post-retrieval and those from oocytes that were mature at the time of retrieval were cytogenetically assessed using a five-color fluorescent in situ fluorescent hybridization assay developed for rhesus macaque chromosomes homologous to human chromosomes 13, 16, 18, X and Y. RESULTS: Blastomeres from 53 embryos were analyzed. Of the 27 embryos that developed from oocytes that were mature at collection, 18 embryos were chromosomally normal (66.7%), while from the 26 embryos that developed from oocytes that matured post-retrieval, only 9 embryos were chromosomally normal (34.6%). CONCLUSIONS: These results indicate that embryos developing from oocytes maturing post-retrieval display high rates of chromosomal abnormalities and have therefore a reduced developmental competence. As a result, the clinical relevance of using immature oocytes that are retrieved after stimulated cycles in human IVF warrants further investigation.

Chromosomal instability in rhesus macaque preimplantation embryos.

OBJECTIVE: To establish a relevant animal model to systematically investigate chromosomal instability in human oocytes and preimplantation embryos. DESIGN: Prospective rhesus monkey IVF study. SETTING: Academic laboratory, Oregon National Primate Research Center and Caribbean Primate Research Center. ANIMAL(S): Young rhesus macaque females. INTERVENTION(S): In vitro produced entire rhesus macaque preimplantation embryos were cytogenetically assessed using a five-color fluorescent in situ hybridization assay developed for rhesus macaque chromosomes homologous to human chromosomes 13, 16, 18, X, and Y, using human bacterial artificial chromosome probes. MAIN OUTCOME MEASURE(S): Chromosomal abnormality rates in preimplantation embryos from young rhesus macaque females were established. RESULT(S): Fifty preimplantation embryos, displaying good morphology and normal development, were analyzed from 11 young rhesus macaque females. Overall, 27 embryos (54%) were normal, 11 embryos (22%) mosaic, 3 embryos (6%) chaotic, 2 embryos (4%) aneuploid, 3 embryos (6%) haploid, and 4 embryos (8%) triploid. CONCLUSION(S): These data indicate that in vitro produced rhesus macaque and human preimplantation embryos exhibit similar numerical chromosomal aberrations. Rhesus macaques appear to be a suitable animal model for investigating the origin of chromosomal instability observed in human preimplantation embryos.

The mitochondrial contribution to stem cell biology.

The distribution and functions of mitochondria in stem cells have not been examined, yet the contributions of these organelles to stem cell viability and differentiation must be vitally important in view of their critical roles in all other cell types. A key role for mitochondria in stem cells is indicated by reports that they translocate in the oocyte during fertilisation to cluster around the pronuclei and can remain in a perinuclear pattern during embryo development. This clustering appears to be essential for normal embryonic development. Because embryonic stem cells are derived from fertilised oocytes, and eventually can differentiate into 'adult' stem cells, it was hypothesised that mitochondrial perinuclear clustering persists through preimplantation embryo development into the stem cells, and that this localisation is indicative of stem cell pluripotency. Further, it was predicted that mitochondrial activity, as measured by respiration and adenosine triphosphate (ATP) content, would correlate with the degree of perinuclear clustering. It was also predicted that these morphological and metabolic measurements could serve as indicators of 'stemness.' This article reviews the distribution and metabolism of mitochondria in a model stem cell line and how this information is related to passage number, differentiation and/or senescence. In addition, it describes mitochondrial DNA deletions in oocytes and embryos that could adversely affect stem cell performance.

Challenges of primate embryonic stem cell research.

Embryonic stem (ES) cells hold great promise for treating degenerative diseases, including diabetes, Parkinson's, Alzheimer's, neural degeneration, and cardiomyopathies. This research is controversial to some because producing ES cells requires destroying embryos, which generally means human embryos. However, some of the surplus human embryos available from in vitro fertilization (IVF) clinics may have a high rate of genetic errors and therefore would be unsuitable for ES cell research. Although gross chromosome errors can readily be detected in ES cells, other anomalies such as mitochondrial DNA defects may have gone unrecognized. An insurmountable problem is that there are no human ES cells derived from in vivo-produced embryos to provide normal comparative data. In contrast, some monkey ES cell lines have been produced using in vivo-generated, normal embryos obtained from fertile animals; these can represent a "gold standard" for primate ES cells. In this review, we argue a need for strong research programs using rhesus monkey ES cells, conducted in parallel with studies on human ES and adult stem cells, to derive the maximum information about the biology of normal stem cells and to produce technical protocols for their directed differentiation into safe and functional replacement cells, tissues, and organs. In contrast, ES cell research using only human cell lines is likely to be incomplete, which could hinder research progress, and delay or diminish the effective application of ES cell technology to the treatment of human diseases.

Duration and temperature of culture medium equilibration affect frequency of blastocyst development.

Hamster 2-cell embryos were cultured in 50 microl drops of chemically defined medium (HECM-9) under oil in 60 mm Petri dishes. In the first experiment, the dishes were equilibrated with 5% O(2) /10% CO(2) /85% N(2) for 2 h either within sealed plastic bags or exposed directly to the same gas mixture in a tissue culture incubator. After culture of embryos for 48 h, there was no difference in development to the blastocyst stage. In the second experiment, the dishes were first equilibrated with 5% O(2) /10% CO(2) / 85% N(2) within sealed plastic bags, (A) at 4 degrees C overnight (16-18 h), or (B) at 37.5 degrees C overnight or (C) at 37.5 degrees C for 2 h. Dishes in treatment A were placed in the incubator at 37.5 degrees C for 2 h next day just before use. Two-cell embryos from a superovulated, mated female were equally distributed among the three treatments, then the dishes were sealed in fresh bags containing the same gas mixture and incubated at 37.5 degrees C for 48 h. This experiment was replicated 13 times with a total of 20 females and 268-275 embryos/treatment. There was no significant difference among the treatments for development to the (combined) morula/blastocyst stages. However, the percentage of blastocysts that developed in culture dishes that had been equilibrated overnight at 37.5 degrees C (treatment B) was significantly lower [50 +/- 14% (SEM)] than in treatments A and C, which were not different from one another (67 +/- 11 and 60 +/- 17% respectively). These results indicate that when culture medium is incubated at 37.5 degrees C overnight, chemical deterioration occurs that is detrimental to embryo development, and that this can be avoided by equilibrating dishes at 4 degrees C overnight, followed by a brief period at 37.5 degrees C to warm the medium before inserting embryos. This finding may have clinical relevance for human embryo culture. The study also demonstrates the utility and advantages of the sealed bag system for embryo culture.

Human ovarian tissue cultures: extracellular matrix composition, coating density and tissue dimensions.

Human ovarian tissue can be successfully cryopreserved for fertility preservation. Optimal use of this approach requires the development of reliable restoration methods, including in-vitro culture of follicles. A culture system has been established, but improvement of the basic handling and techniques is necessary. Ovarian biopsies were collected from 33 women, cut into small pieces and cultured for 7-14 days on an extracellular matrix. Three separate studies investigated tissue dimensions (slices and cubes), coating density of extracellular matrix (diluted, thin and thick), and different extracellular matrix compositions (regular Matrigel, growth factor reduced Matrigel and laminin). Initial recruitment of primordial follicles and reduction in follicle viability was observed in all cultures compared with uncultured tissue. After 7 days of culture, more viable follicles were present in the cubed tissue, which also showed significant activation of growth, observed in tissue slices only after 14 days of culture. A diluted coating of Matrigel supported a greater proportion of viable follicles in 7-day cultures, whereas composition of the extracellular matrix had no effect. Human ovarian follicles can grow and develop in vitro within cortical tissue, and may benefit from culture as cubes on diluted Matrigel. This technique may provide a solution to the successful recovery and growth of follicles from frozen human ovarian tissue even though it will take time and much more optimization before it can be used in clinical practice.

17Beta-estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys.

BACKGROUND: Effects of 17beta-estradiol and progesterone on rhesus monkey oocyte maturation in vitro were evaluated by embryo development subsequent to IVF. METHODS AND RESULTS: In experiment 1, immature cumulus-oocyte complexes collected from unstimulated adult females during the non-breeding season were matured in modified medium CMRL-1066 containing various combinations of gonadotrophins (FSH + LH), estradiol and/or progesterone. Formation of morulae and blastocysts was greatest in oocytes matured in medium containing estradiol and/or progesterone, with or without gonadotrophins (morula 38-46%, blastocyst 14-20%) than in control oocytes matured without estradiol or progesterone (morula 14%, blastocyst 0%). In experiment 2, cumulus-oocyte complexes from unstimulated prepubertal female monkeys were matured in medium with gonadotrophins, estradiol or progesterone. The best development to the morula stage was obtained with oocytes matured with gonadotrophins and estradiol or gonadotrophins and progesterone (43 and 25 morulae, respectively), while control oocytes matured with gonadotrophins but without steroid hormones gave the poorest morula developmental response (12%). However, there was no difference in blastocyst development across all groups (0-3%). CONCLUSIONS: These results demonstrate that during rhesus monkey oocyte maturation in vitro: (i) estradiol or progesterone can improve oocyte developmental competence; (ii) immature oocytes from prepubertal versus adult females have differential responses to challenge with estradiol or progesterone.

Blastocyst development after intergeneric nuclear transfer of mountain bongo antelope somatic cells into bovine oocytes.

Intergeneric embryos were constructed by nuclear transfer using Mountain Bongo antelope somatic cells fused with enucleated bovine oocytes and their subsequent development in vitro was investigated. After two to six passages, starved or non-starved skin fibroblast cells were used as donor nuclei. In vitro matured bovine oocytes were enucleated by squeezing the first polar body and surrounding cytoplasm through a slit in the zona pellucida. After injection of a somatic cell into the perivitelline space, couplets were fused electrically and activated chemically, then subjected to different embryo culture treatments. Serum starvation had no effect on the frequency of cleavage to two cells or on development to the blastocyst stage in either sequential hamster embryo culture medium (HECM)-6/TCM-199 + serum or HECM-9/TC-199 + serum, or modified synthetic oviduct fluid (mSOF) culture medium. When couplets from non-starved donor nuclei were cultured, the frequency of cleavage (66 +/- 8% vs. 44 +/- 5%), development to >/=9 cells (46 +/- 6% vs. 24 +/- 4%), and formation of blastocysts (24 +/- 5% vs. 11 +/- 2%) were all significantly higher (p < 0.05) in the HECM-6 medium than in mSOF medium. In conclusion, bovine oocytes can support blastocyst development after intergeneric fusion with bongo fibroblasts. This technique could potentially be used as an alternative to using scarce bongo oocytes in attempts to propagate these endangered animals.

Early history of in vitro fertilization.

Although in vitro fertilization (IVF) is used widely for a variety of purposes, it is often not appreciated how this technology was developed. A large number of experiments beginning in 1878 contributed to the first successful reports of IVF over 75 years later. The discovery of sperm capacitation in 1951 was central to the development of IVF technology, and it was rapidly followed by the first convincing reports of IVF in several species. The ability to fertilize oocytes in vitro has allowed major advances to be made into understanding the mechanisms involved in fertilization and early development, and IVF now supports reproductive biotechnology in animals and in humans. This article is a historical review of key experiments that helped to provide the basis for present day IVF procedures, placed into context with current practice.

Features associated with reproductive ageing in female rhesus monkeys.

BACKGROUND: The specific aims were to determine the effects of maternal age on the meiotic and developmental competence of oocytes and incidence of chromosomal anomalies in oocytes from a population of fertile rhesus monkeys. METHODS: Monkeys were divided into two age groups (4-15 and 16-26 years of age) and underwent ovarian stimulation for collection of oocytes. RESULTS: In the older, compared with younger, monkeys, serum basal concentrations of FSH were elevated (P < 0.05), peak concentrations of estradiol during a stimulation cycle were diminished (P < 0.05), and mean numbers of oocytes retrieved following ovarian stimulation were markedly (P < 0.05) reduced. There were no significant maternal age-related impairments in oocyte maturation, fertilization or blastocyst development. Both abnormal numbers of whole chromosomes, as well as free chromatids, were detected in a limited number of rhesus oocytes. CONCLUSIONS: Similarities between female rhesus monkeys and women in several features associated with reproductive ageing, in conjunction with our ability to perform IVF and other assisted reproductive techniques in monkeys, demonstrate the suitability of these animals for studies on human reproductive ageing and maternal age-related infertility. Although maternal age-related impairments in oocytes were not evident prior to implantation, further studies may reveal more subtle impairments, manifested during post-implantation development.

Biochemical and biological properties of urinary follicle-stimulating hormone (FSH) from the rhesus monkey (Macaca mulatta).

Follicle-stimulating hormone (FSH) is routinely used for the induction of superovulation in women. Homologous gonadotropin preparations that could be used for reproductive studies in macaques would have valuable research applications. Accordingly, we set out to characterize the physical and biological characteristics of urinary FSH (UFSH) in the ovariectomized rhesus monkey. In urine from 7 monkeys, concentrations of bioactive FSH ranged from 16 to 57 µg/1, relative to cynFSH-RPI (NIDDK). UFSH was contrasted to pituitary FSH (PFSH) by non-reducing SDS-polyacrylamide gel electrophoresis (PAGE), native disc PAGE, and FPLC chromatofocusing. The apparent molecular weights of UFSH and PFSH are similar (approximately 35 kD); however, UFSH is more negatively charged and demonstrates a lower overall isoelectric (pl) range than PFSH. The bioactivity of UFSH was assessed by the stimulation of aromatase activity in cultured Sertoli cells and by induction of follicular maturation in hamsters. Two fractions of pituitary FSH, which differed in isoelectric properties, were obtained by chromatofocusing. The in vivo biological activity of FSH-A (acidic, pl 3.8-4.6) and UFSH (pl 3.5-4.5) were similar, but greater than FSH-B (basic, pl 4.6-5.5). These results support the hypothesis that heavily sialylated, low pl FSH expresses high in vivo bioactivity. This may reflect the well-known effect of sialic acid in prolonging the circulating half-life of glycoproteins. Thus, the quality and quantity of FSH present in ovariectomized rhesus monkey urine indicates that this may be a useful source for the preparation of enriched hormone preparations. © 1992 Wiley-Liss, Inc.

RECENT PROGRESS IN THE STUDY OF EARLY EVENTS IN MAMMALIAN FERTILIZATION.

During the past 25 years, great advances have been made in understanding the physiology, morphology and biochemistry of fertilization in invertebrate animal species. In contrast to this situation, there is a paucity of knowledge pertaining to mammalian fertilization. Major areas in which information is lacking are the nature of changes undergone by spermatozoa in preparation for fertilization, and the mechanisms involved in sperm penetration of the egg investments. The present state of knowledge of these events is outlined, and the weaknesses of some current concepts are evaluated. Fertilization of mammalian eggs in vitro seems an attractive method for studying gamete interaction, but experience has shown that numerous problems are associated with this technique. As a result, the information on mammalian fertilization that has been derived from studies conducted in vitro has fallen considerably short of expectations; some factors contributing to this discrepancy are described. Recent findings concerning the regulation of sperm motility and fertilizing ability seem to have considerable significance for mammalian fertilization in vivo and in vitro. These findings have been utilized to refine existing procedures; fertilization of hamster eggs in vitro has now been accomplished in the presence of numbers of spermatozoa comparable to those believed to be present at the site and time of fertilization in vivo. It is anticipated that this improved technique, by more closely approximating the physiological situation, will substantially assist the derivation of useful information from in vitro fertilization studies.