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Reproduction in farm animals

Thyroid hormone is a critical regulator of growth, development, and metabolism in virtually all tissues, and altered thyroid status affects many organs and systems. Although for many years testis has been regarded as a thyroid hormone unresponsive organ, it is now evident that thyroid hormone plays an important role in testicular development and function. A considerable amount of data show that thyroid hormone influences steroidogenesis as well as spermatogenesis. The involvement of tri-iodothyronine (T(3)) in the control of Sertoli cell proliferation and functional maturation is widely accepted, as well as its role in postnatal Leydig cell differentiation and steroidogenesis. The presence of thyroid hormone receptors in testicular cells throughout development and in adulthood implies that T(3) may act directly on these cells to bring about its effects. Several recent studies have employed different methodologies and techniques in an attempt to understand the mechanisms underlying thyroid hormone effects on testicular cells. The current review aims at presenting an updated picture of the recent advances made regarding the role of thyroid hormones in male gonadal function.

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The role of thyroid hormone in testicular development and function.

Size control is fundamental in tissue development and homeostasis1,2. Although the role of cell proliferation in these processes has been widely studied, the mechanisms that control embryo size-and how these mechanisms affect cell fate-remain unknown. Here we use the mouse blastocyst as a model to unravel a key role of fluid-filled lumen in the control of embryo size and specification of cell fate. We find that there is a twofold increase in lumenal pressure during blastocyst development, which translates into a concomitant increase in cell cortical tension and tissue stiffness of the trophectoderm that lines the lumen. Increased cortical tension leads to vinculin mechanosensing and maturation of functional tight junctions, which establishes a positive feedback loop to accommodate lumen growth. When the cortical tension reaches a critical threshold, cell-cell adhesion cannot be sustained during mitotic entry, which leads to trophectoderm rupture and blastocyst collapse. A simple theory of hydraulically gated oscillations recapitulates the observed dynamics of size oscillations, and predicts the scaling of embryo size with tissue volume. This theory further predicts that disrupted tight junctions or increased tissue stiffness lead to a smaller embryo size, which we verified by biophysical, embryological, pharmacological and genetic perturbations. Changes in lumenal pressure and size can influence the cell division pattern of the trophectoderm, and thereby affect cell allocation and fate. Our study reveals how lumenal pressure and tissue mechanics control embryo size at the tissue scale, which is coupled to cell position and fate at the cellular scale.

Hydraulic control of mammalian embryo size and cell fate

Processing and characterization of silk sericin from Bombyx mori and its application in biomaterials and biomedicines.

The present invention relates to the discovery that biocompatible anionic polymers can effectively inhibit fibrosis, scar formation, and surgical adhesions. The invention is predicated on the discovery that anionic polymers effectively inhibit invasion of cells associated with detrimental healing processes, and in particular, that the effectiveness of an anionic polymer at inhibiting cell invasion correlates with the anionic charge density of the polymer. Thus the present invention provides a large number of materials for use in methods of inhibiting fibrosis and fibroblast invasion. Anionic polymers for use in the invention include but are not limited to natural proteoglycans, and the glycosaminoglycan moieties of proteoglycans. Additionally, anionic carbohydrates and other anionic polymers may be used. The anionic polymers dextran sulfate and pentosan polysulfate are preferred. In a more preferred embodiment, dextran sulfate, in which the sulfur content is greater than about 10% by weight, may be used. In a more preferred embodiment, the average molecular weight is about 40,000 to 500,000 Daltons. The present invention provides compositions and methods to inhibit fibrosis and scarring associated with surgery. The invention further provides compositions and methods to inhibit glial cell invasion, detrimental bone growth and neurite outgrowth. In a preferred embodiment, the inhibitory compositions further comprise an adhesive protein.

Methods and compositions based on inhibition of cell invasion and fibrosis by anionic polymers

Contraction has been observed in cultured blastocysts of many mammals, but little is known about the features of the contraction and its physiological role in blastocysts. The author analyzed contractions of a large number of cultured mouse blastocysts by time-lapse videomicrography. The results revealed that blastocysts repeated contractions of different degrees during the expanded stage from 10 h after blastocoel formation, and that the number of contractions was greater during the hatching period than in the periods pre- and post-hatching. The results also showed that the time needed for both contraction and re-expansion to the size before contraction tended to lengthen in blastocysts severely contracted. It was inferred that contractions of blastocysts occur physiologically in relation to myosin light chain kinase, but not due to an increase in permeability between trophectoderm cells in association with their division, or the influence of culture. Furthermore, it was inferred that re-expansion of contracted blastocysts occurs due to active transport and accumulation of Na(+) from the trophectoderm cells into blastocoelic fluid as a result of the action of Na(+)/K(+)-ATPase activated in the membrane of trophectoderm cells. Our results suggested that contractions are also present in blastocysts developed in vivo, and that weak contractions (less than 20% volume reduction) play an important role in hatching, whereas strong contractions (20% or more volume reduction) have the effect of inhibiting hatching. From our results on contractions of various blastocysts, it seems possible to evaluate the developmental ability of embryos, i.e. embryo quality, based on contractions of blastocysts.

Time-lapse videomicrographic analyses of contractions in mouse blastocysts.

Production of hyaluronan (hyaluronic acid: HA) was demonstrated in denuded mouse oocytes (DOs) by the enzyme-linked immunosorbent assay, and the role of HA in enlargement of the perivitelline space in the oocytes was examined. The incidence of polyspermy following insemination was also observed in DOs in which HA synthesis was inhibited. HA was not detected in culture medium containing DOs immediately after collection. After culture for 7 h, 4.75 pg of HA per DO was detected in the medium, and the mean amount of HA significantly increased to 20.78 pg 14 h after culture. When DOs were cultured in medium containing 0.25 mM 4-methylumbelliferone (MU), an inhibitor of HA synthase, the mean amount of HA in the culture medium with DOs was 8.61 pg, which was significantly smaller than the amount in the control medium with non-treated DOs (21.59 pg). The mean size of the perivitelline space in oocytes cultured with cumulus cells (5.40 mum) did not differ from that (5.08 mum) of DOs. The mean size of the perivitelline space was significantly smaller in the MU-treated DOs (3.58 mum) than in the control DOs (4.65 mum). The fertilization rate did not differ between the MU-treated DOs (84.9%) and control DOs (81.0%), whereas the incidence of polyspermy was significantly higher in the MU-treated DOs (13.3%) compared with the control DOs (2.1%). These findings clarified that the HA involved in enlargement of the perivitelline space in oocytes is synthesized and secreted by the oocytes themselves. They also suggest that there is a close relationship between the size of perivitelline space and the incidence of polyspermy in mouse oocytes.

Amount of Hyaluronan Produced by Mouse Oocytes and Role of Hyaluronan in Enlargement of the Perivitelline Space

Morphological changes of cultured bovine blastocysts during hatching were observed using time-lapse videomicrography in order to investigate the patterns of the hatching process that occurred in the blastocysts and to determine whether the hatching patterns differed between blastocysts developed from fresh and cryopreserved embryos. Compacted morulae (CMs) were collected from superovulation-treated Japanese Black and Holstein dairy cattle and cultured in a medium in a CO(2) culture chamber equipped with an inverted microscope at 38.5 C. Images of resultant blastocysts during the period from blastocoel formation to completion of hatching were taken at 4-sec intervals by a CCD color camera connected to an inverted microscope and recorded by a time-lapse video cassette recorder. In blastocysts developed from fresh CMs, hatching was found to begin with protrusion of trophectoderm cells from zonae pellucidae at the expanded stage. Protrusion of the cells occurred in any site of the trophectoderm. After protrusion, a large or small slit was formed in the zona pellucida in all blastocysts as a result of blastocyst expansion or enlargement of the protrusion. Then, blastocysts completely escaped from the zona pellucida through the slit in the state of expansion. From these findings, the hatching patterns of cattle blastocysts could be classified into 5 types. In blastocysts developed from frozen-thawed CMs, the hatching pattern and length of time needed for hatching are similar to those in blastocysts developed from fresh CMs. In addition, the pregnancy rate of recipients following transfer of frozen-thawed CMs (52.4%) did not differ from that of recipients following transfer with fresh CMs (58.3%). These results suggested that the quality of frozen-thawed cattle embryos is comparable to that of fresh embryos and that there could be a relationship between the hatching pattern of blastocysts and the viability of embryos after transfer.

Time-Lapse Videomicrographic Observations of Blastocyst Hatching in Cattle

In mouse oocytes soon after collection, perivitelline space could be seen in about half of them and its mean size was 0.28 μm. After 6 hrs of in vivo and in vitro maturation, perivitelline space appeared in all oocytes. In oocytes in the process of maturation in vivo the mean size of perivitelline space significantly increased 6 hrs after hCG injection and reached 7.00 μm at 14 hrs after the hCG injection. On the other hand, perivitelline space in cultured oocytes enlarged significantly at 2 hrs culture. Thereafter, perivitelline space in oocytes continued to enlarge with culture time and reached 5.40 μm at 14 hrs of maturation culture. The size of perivitelline space in oocytes 14 hrs after the hCG injection was significantly larger than that in oocytes cultured for 14 hrs. The fertilization rates of oocytes matured in vivo (85%) did not differ from that of oocytes matured in vitro (80%). However, the incidence of polyspermy in oocytes matured in vivo was 4%, which was significantly lower than the 16% of oocytes matured in vitro. These findings suggest there could be a relationship between the size of the perivitelline space and the incidence of polyspermy in mouse oocytes.

Size of Perivitelline Space and Incidence of Polyspermy in Mouse Oocytes Matured In Vivo and In Vitro

Congenital hypothyroid mutant male rdw rats have enlarged testes in adulthood with dwarfism accompanied by infertility. To explain how rdw rats acquire enlarged testes in adulthood, we compared age-matched normal (N) rats at various developmental stages for blood levels of hormones, thyroxine (T4), follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T), and investigated whether T4 therapy (rdw+T4) from 3 weeks of age (w) until adulthood could induce recovery of fertility in rdw rats, as well as how rdw+T4 affected hormonal patterns. Testes weights of rdw rats were higher than those of N rats at 19 w in adulthood though it was low during development. Serum T4 values in rdw rats were markedly lower than those in N rats but steadily increased up to 19 w. The serum FSH values in rdw rats were lower than those in N rats at all ages, and neither serum LH nor T value was significantly different at any age. The testes weight of rdw+T4 rats was significantly higher than that of N rats at 13 w with recovered growth, and was higher than that of rdw rats at 19 w. When they were mated with proestrous females after 16 w, all females became pregnant and gave birth to a normal number of pups. The T4 and FSH values of rdw+T4 rats were significantly higher than those in rdw rats, but similar to those in N rats in adulthood. The results suggest that even low levels of circulating thyroid hormone (TH) in rdw rats stimulate the development of their testes, probably through Sertoli cells, resulting in the enlarged adult testes without fertility, and that a sufficient circulating TH level from the immature stage plays a pivotal role in restoring mating activity, probably through FSH-mediated action towards adulthood.

Sueo Niimura

Papers

Time-lapse videomicrographic analyses of contractions in mouse blastocysts.

Amount of Hyaluronan Produced by Mouse Oocytes and Role of Hyaluronan in Enlargement of the Perivitelline Space

Time-Lapse Videomicrographic Observations of Blastocyst Hatching in Cattle

Size of Perivitelline Space and Incidence of Polyspermy in Mouse Oocytes Matured In Vivo and In Vitro

Developmental Hormonal Profiles in rdw Rats with Congenital Hypothyroidism Accompanying Increased Testicular Size and Infertility in Adulthood